Cancers

14 pages, 11192 KiB

Open AccessArticle

Crosstalk between ERα and Receptor Tyrosine Kinase Signalling and Implications for the Development of Anti-Endocrine Resistance

by Rugaia Z. Montaser and Helen M. Coley

Cancers 2018, 10(6), 209; https://doi.org/10.3390/cancers10060209 - 20 Jun 2018

Cited by 10 | Viewed by 3931

Abstract

Although anti-endocrine therapies have significantly advanced the treatment of breast cancer, they pose the problem of acquired drug resistance. The oestrogen receptor (ER)-expressing breast cancer cell lines MCF-7 and T47D alongside their in vitro derived resistant counterparts MCF-7-TR (tamoxifen-resistant) and T47D-FR (fulvestrant-resistant) showed [...] Read more.

Although anti-endocrine therapies have significantly advanced the treatment of breast cancer, they pose the problem of acquired drug resistance. The oestrogen receptor (ER)-expressing breast cancer cell lines MCF-7 and T47D alongside their in vitro derived resistant counterparts MCF-7-TR (tamoxifen-resistant) and T47D-FR (fulvestrant-resistant) showed dual resistance to fulvestrant and tamoxifen in the presence of upregulated HER1 and HER2 growth factor receptors. Our study demonstrated that tamoxifen resistance and fulvestrant resistance are associated with collateral sensitivity to the tyrosine kinase inhibitors (TKIs) lapatinib (p < 0.0001) and afatinib (p < 0.0001). Further, we found that over time, the TKIs reactivated ERα protein and/or mRNA in tamoxifen- and fulvestrant-resistant cells. Combinations of anti-endocrine agents with afatinib gave rise to significantly enhanced levels of apoptosis in both T47D-FR and MCF-7-TR in a synergistic manner versus additive effects of agents used singly. This was associated with p27^kip1 induction for anti-endocrine-resistant cells versus parental cells. Our data supports the use of combination treatment utilising dual HER1/2 inhibitors in breast cancer patients showing resistance to multiple anti-endocrine agents. Full article

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Assessment of tamoxifen (upper panels) and fulvestrant (lower panels) sensitivity in breast cancer cell lines. An MTT assay was used to measure sensitivity to tamoxifen and a clonogenic assay was used for assessment of fulvestrant sensitivity (n ≥ 4). Dose–response curves were fitted using the Prism (version 6) program. IC50 values indicating the levels of drug resistance are shown by bar graphs using a paired t-test: *** p < 0.001; **** p < 0.0001. Full article ">Figure 2
Levels of HER expression in breast cancer cell lines with acquired resistance to anti-oestrogen therapy. Representative Western blot data obtained from whole-cell lysates (A) separated by SDS-PAGE and immunoblotted onto PVDF membranes before immunodetection (n = 4). The head and neck cancer HN5 cell line HER1 + ve and breast cancer SKBR3 cell line HER2 + ve were used as positive controls. (B) Expression levels of parental cells (set at 1.0) were compared with the resistant variant using the following paired t-test levels of statistical significance for densitometric scans: * p < 0.05; ** p < 0.001; *** p < 0.0001. Full article ">Figure 3
Sensitivity of breast cancer cell lines with acquired resistance to anti-oestrogen therapy to tyrosine kinase inhibitors (TKIs), assessed by MTT assay (n ≥ 4), (A) MCF-7 and (B) T47D cell lines. Dose–response curves were fitted using the Prism (version 6) program. Data show a broad collateral sensitivity of anti-endocrine-resistant cells to lapatinib, gefitinib, and afatinib. See <a href="#sec2dot3-cancers-10-00209" class="html-sec">Section 2.3</a> for statistical analyses. Full article ">Figure 4
HER1 and HER2 expression with and without tyrosine kinase inhibitor (TKI) treatment in parental MCF-7 and MCF-7-TR (tamoxifen-resistant) and T47D and T47D-FR (fulvestrant-resistant) breast cancer cell lines using confocal fluorescence microscopy. Cells were treated with 5 µM lapatinib and incubated for 48 h. TO-PRO-3 (blue) was used to label nuclei, and HER1 (A) and HER2 (B) antibody reactions were conjugated to Alexa-Fluor 488 secondary antibody. Images are representative of at least three separate experiments. Settings for the microscope were maintained as consistently as possible from one experiment to another. Full article ">Figure 5
(A) Levels of p27kip1 expression in drug-sensitive and -resistant MCF-7 and T47D breast cancer cell lines in the absence or presence of the 5 µM lapatinib and anti-oestrogen therapy, with densitometry levels shown (B). (C) Constitutive expression of Skp2 in breast cancer cell lines. Representative Western blot data obtained from whole-cell lysates separated by SDS-PAGE, immunoblotted onto PVDF membranes before immunodetection (n = 3). Expression levels were set as 1.0 (control cells) using densitometric analysis and are then shown in response to the various drug treatments relative to each control: * p < 0.05; ** p < 0.001. Full article ">Figure 6
Oestrogen receptor-α (ERα) re-activation following treatment of anti-oestrogen-resistant breast cancer cell lines with lapatinib. (A,B) Representative Western blots of ERα and activated pERα at time points up to 72 h for MCF-7 and T47D cell lines, respectively. Representative Western blot data obtained from whole-cell lysates (upper panel) separated by SDS-PAGE, immunoblotted onto PVDF membranes before immunodetection (n = 3). (C,D) Levels of fold change in ESR mRNA as measured by qPCR and corrected to GAPDH as the housekeeping gene, using the ΔΔCt equation. Expression in MCF-7 (C) and T47D (D) parental cell line control (untreated) values were normalised to 1.0. Statistical analysis was performed using a two-tailed t-test: * p < 0.05; ** p < 0.01. Full article ">Figure 7
Flow cytometry data using annexin V (conjugated to fluoroscein isothiocyanate—FITC) (FL1) and propidium iodide (PI) (FL3) showing the effect of afatinib in combination with anti-oestrogens (48 h treatment) in MCF-7 parent and MCF-7-TR (tamoxifen-resistant) cell lines (A), and in T47D parent and T47D-FR (fulvestrant-resistant) cell lines (B) using the lowest dose levels of drugs (2.5 µM afatinib, 5.0 µM tamoxifen, or 0.5 µM fulvestrant). The lower left-hand quadrant of each representative data point cytogram represents the live cell population (FITC- and PI-negative). The percentages displayed indicate the live cell component in the given examples; (C) shows the combination index (CI) values obtained for the various drug treatments at three different levels (afatinib: 2.5 µM, 5.0 µM, and 10.0 µM; tamoxifen: 5 µM, 10.0 µM, and 20.0 µM; fulvestrant: 0.5 µM, 1.0 µM, and 2.0 µM). Comparisons between parent and resistant cell line CI values were made by two-way ANOVA with Bonferroni correction: * p < 0.001; ** p < 0.0001. (D) The various phases of the cell cycle according to drug treatment; data shown are representative of repeat experiments (n = 4). See <a href="#sec2dot8-cancers-10-00209" class="html-sec">Section 2.8</a> for statistical analysis. Full article ">

9 pages, 219 KiB

Open AccessConference Report

Innovative Technologies Changing Cancer Treatment

by Sara Charmsaz, Maria Prencipe, Maeve Kiely, Graham P. Pidgeon and Denis M. Collins

Cancers 2018, 10(6), 208; https://doi.org/10.3390/cancers10060208 - 19 Jun 2018

Cited by 21 | Viewed by 4834

Abstract

Conventional therapies for cancer such as chemotherapy and radiotherapy remain a mainstay in treatment, but in many cases a targeted approach is lacking, and patients can be vulnerable to drug resistance. In recent years, novel concepts have been emerging to improve the traditional [...] Read more.

Conventional therapies for cancer such as chemotherapy and radiotherapy remain a mainstay in treatment, but in many cases a targeted approach is lacking, and patients can be vulnerable to drug resistance. In recent years, novel concepts have been emerging to improve the traditional therapeutic options in cancers with poor survival outcomes. New therapeutic strategies involving areas like energy metabolism and extracellular vesicles along with advances in immunotherapy and nanotechnology are driving the next generation of cancer treatments. The development of fields such as theranostics in nanomedicine is also opening new doors for targeted drug delivery and nano-imaging. Here we discuss the use of innovative technologies presented at the Irish Association for Cancer Research (IACR) Annual Meeting, highlighting examples of where new approaches may lead to promising new treatment options for a range of cancer types. Full article

19 pages, 1433 KiB

Open AccessEditor’s ChoiceReview

Hypersialylation in Cancer: Modulation of Inflammation and Therapeutic Opportunities

by Emily Rodrigues and Matthew S. Macauley

Cancers 2018, 10(6), 207; https://doi.org/10.3390/cancers10060207 - 18 Jun 2018

Cited by 142 | Viewed by 10122

Abstract

Cell surface glycosylation is dynamic and often changes in response to cellular differentiation under physiological or pathophysiological conditions. Altered glycosylation on cancers cells is gaining attention due its wide-spread occurrence across a variety of cancer types and recent studies that have documented functional [...] Read more.

Cell surface glycosylation is dynamic and often changes in response to cellular differentiation under physiological or pathophysiological conditions. Altered glycosylation on cancers cells is gaining attention due its wide-spread occurrence across a variety of cancer types and recent studies that have documented functional roles for aberrant glycosylation in driving cancer progression at various stages. One change in glycosylation that can correlate with cancer stage and disease prognosis is hypersialylation. Increased levels of sialic acid are pervasive in cancer and a growing body of evidence demonstrates how hypersialylation is advantageous to cancer cells, particularly from the perspective of modulating immune cell responses. Sialic acid-binding receptors, such as Siglecs and Selectins, are well-positioned to be exploited by cancer hypersialylation. Evidence is also mounting that Siglecs modulate key immune cell types in the tumor microenvironment, particularly those responsible for maintaining the appropriate inflammatory environment. From these studies have come new and innovative ways to block the effects of hypersialylation by directly reducing sialic acid on cancer cells or blocking interactions between sialic acid and Siglecs or Selectins. Here we review recent works examining how cancer cells become hypersialylated, how hypersialylation benefits cancer cells and tumors, and proposed therapies to abrogate hypersialylation of cancer. Full article

(This article belongs to the Special Issue Inflammation and Cancer)

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Figure 1

Figure 1
Hypersialylation in cancer: causes and effects. Elevated levels of sialic acid on transformed cells can be driven by at least three different mechanisms. Hypersialylation on cancer cells can promote tumor development and survival in a many of different ways but one key mechanism is through modulating immune cell responses and in particular those immune cells types involved in modulating the inflammatory environment in tumors. Full article ">Figure 2
Roles for hypersialylation on cancer in modulating Siglecs on immune cells. (a) Siglec-7 (and potentially Siglec-9) on NK cells can be drawn into an immunological synapse formed with a cancer cell to inhibit an activatory receptor (e.g., CD16), thereby inhibiting NK cell-mediated killing; (b) Siglec-9 on neutrophils can be engaged by hypersialylation of cancer cells and prevent neutrophil-mediated killing of cancer cells through an unknown activatory receptor and ligand on the cancer cells; (c) Siglec-9 on macrophages can promote or inhibit skewing of macrophages to a tumor-associated or tumor-promoting phenotype through engagement of sialic acid-containing glycans on cancer cells; (d) Siglec-15 on macrophages pairs with the adapter protein Dap12 to activate cellular signaling in response to sialic acid ligands on cancer cells, thereby inducing TGF-β production; (e) CD33 (Siglec-3) on myeloid-derived suppressor cells (MDSCs) has been shown to modulate inflammatory responses through modulating TLR4. Full article ">Figure 3
Therapeutic strategies for modulating sialic acid expression and interactions between sialic acid and sialic acid-binding receptors. (Upper left portion of cell): delivery of 3F-Neu5Ac to cancer cells to decrease sialic acid expression. 3F-Neu5Ac is typically delivered in its peracetylated form to penetrate the cell membrane. Once inside the cell, 3F-Neu5Ac is converted into CMP-3F-Neu5Ac and blocks the actions of STs in the secretory pathway. (Upper right portion of cell): blocking antibodies that abrogate the interaction between sialic acid on cancer cells and sialic acid-binding receptors on immune cells, such as Siglecs, have the potential of being a new class of immune checkpoint inhibitor. (Lower portion of the cell): antibody directed to the tumor, such as anti-HER2 to target breast cancer cells, have been conjugated to neuraminidase to cleave the sialic acid residues on the cancer cells. Full article ">

18 pages, 2693 KiB

Open AccessArticle

Constitutive Activation of STAT3 in Myeloma Cells Cultured in a Three-Dimensional, Reconstructed Bone Marrow Model

by Yung-Hsing Huang, Ommoleila Molavi, Abdulraheem Alshareef, Moinul Haque, Qian Wang, Michael P. Chu, Christopher P. Venner, Irwindeep Sandhu, Anthea C. Peters, Afsaneh Lavasanifar and Raymond Lai

Cancers 2018, 10(6), 206; https://doi.org/10.3390/cancers10060206 - 16 Jun 2018

Cited by 20 | Viewed by 5601

Abstract

Malignant cells cultured in three-dimensional (3D) models have been found to be phenotypically and biochemically different from their counterparts cultured conventionally. Since most of these studies employed solid tumor types, how 3D culture affects multiple myeloma (MM) cells is not well understood. Here, [...] Read more.

Malignant cells cultured in three-dimensional (3D) models have been found to be phenotypically and biochemically different from their counterparts cultured conventionally. Since most of these studies employed solid tumor types, how 3D culture affects multiple myeloma (MM) cells is not well understood. Here, we compared MM cells (U266 and RPMI8226) in a 3D culture model with those in conventional culture. While the conventionally cultured cells were present in single cells or small clusters, MM-3D cells grew in large spheroids. We discovered that STAT3 was the pathway that was more activated in 3D in both cell lines. The active form of STAT3 (phospho-STAT3 or pSTAT3), which was absent in MM cells cultured conventionally, became detectable after 1–2 days in 3D culture. This elevated pSTAT3 level was dependent on the 3D environment, since it disappeared after transferring to conventional culture. STAT3 inhibition using a pharmacological agent, Stattic, significantly decreased the cell viability of MM cells and sensitized them to bortezomib in 3D culture. Using an oligonucleotide array, we found that 3D culture significantly increased the expression of several known STAT3 downstream genes implicated in oncogenesis. Since most primary MM tumors are naturally STAT3-active, studies of MM in 3D culture can generate results that are more representative of the disease. Full article

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Figure 1

Figure 1
MM cells exhibit different appearances and growth patterns in conventional culture versus in 3D culture. (A) The morphology of U266 and RPMI8226 cells in conventional or 3D culture after 6 days was examined by phase contrast microscopy. Images were taken at 100X magnification. A scale bar equivalent to 20 µm is included in each graph; (B) The growth of U266 and RPMI8226 cells in conventional (blue bars) or 3D cultures (orange bars) was measured by the trypan blue exclusion assay at various time points. Fold changes of total viable cells were normalized to the cell number on day 0 (2.5 × 105 cells). The error bars represent standard deviation from a triplicate experiment, * p < 0.05, n.s. not significant, Student’s t-test. Full article ">Figure 2
MM cells cultured in 3D acquire STAT3 activity. (A) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; (B) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; (C) The DNA binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); (D) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; (E) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot. Full article ">Figure 3
Acquired STAT3 activity in MM cells diminished upon transfer from 3D to conventional culture. The STAT3 activity in U266 and RPMI8226 cells before and after transfer from 3D culture by Western blot analysis of pSTAT3 level. U266 and RPMI8226 were pre-cultured in 3D culture for 2 days and 1 day prior to transfer to reach a substantial pSTAT3 level, respectively. β-actin was probed as a loading control. 2.5 × 105 cells were seeded initially. Full article ">Figure 4
MM cells cultured in 3D are more sensitive to STAT3 inhibition. CETSA of (A) U266 and (B) RPMI8226 cells in conventional or 3D culture after 1 h of Stattic treatment. Vinculin was blotted as a loading control. The STAT3/vinculin ratios were quantified using ImageJ and shown on the right. Error bars represent the standard deviation from two independent experiments; (C) The effect of STAT3 inhibition on cell viability of U266 and RPMI8226 cells in conventional or 3D culture. The cells were treated with Stattic for 24 h. Cell viability was measured by MTS assay and normalized to cells with no Stattic treatment. The error bars represent standard deviation from a triplicate experiment, * p < 0.05, Student’s t-test; (D) The effect of STAT3 inhibition on apoptosis in U266- and RPMI8226-3D cells. The cells were treated with Stattic for 24 h and stained with an apoptotic marker Annexin V. The percentage of Annexin V-positive cells was analyzed by flow cytometry; (E) The expression levels of two apoptotic markers, cleaved PARP and cleaved caspase 3, in U266- and RPMI8226-3D cells after 24 h of Stattic treatment were examined by Western blot analysis. β-actin was probed as a loading control. For all the experiments above, U266 and RPMI8226 cells were cultured for 2 and 1 days before the Stattic treatment to reach a substantial pSTAT3 level, respectively. 2.5 × 105 cells were seeded initially. Full article ">Figure 5
STAT3 inhibition in MM-3D cells sensitizes them to bortezomib. Cell viability of (A) U266- and (B) RPMI8226-3D cells was measured after treatment with Stattic, bortezomib (BTB) or both for 48 h. U266 and RPMI8226 were pre-cultured in 3D for 2 days and 1 day before drug treatment to reach a substantial pSTAT3 level, respectively. Cell viability was measured by MTS assay and normalized to the cell viability of untreated cells. 2.5 × 105 cells were seeded initially. The error bars represent standard deviation from a triplicate experiment, ** p < 0.001, Student’s t-test. Full article ">Figure 6
3D culture changes the gene expression in MM cells. Quantitative RT-PCR of LPL, ANGPT2, DDIT3 and CA9 mRNA levels in U266 cells in conventional culture (2D) or day 1 to 4 in 3D culture. 2.5 × 105 cells were seeded initially. The primers used for each gene are shown in Materials and Methods. The error bars represent standard deviation from a triplicate experiment, n.s. not significant and ** p < 0.001 compared to 2D, one-way ANOVA with Dunnett’s multiple t-test. Full article ">

18 pages, 2686 KiB

Open AccessArticle

Oncolytic Reovirus and Immune Checkpoint Inhibition as a Novel Immunotherapeutic Strategy for Breast Cancer

by Ahmed A. Mostafa, Daniel E. Meyers, Chandini M. Thirukkumaran, Peter J. Liu, Kathy Gratton, Jason Spurrell, Qiao Shi, Satbir Thakur and Don G. Morris

Cancers 2018, 10(6), 205; https://doi.org/10.3390/cancers10060205 - 15 Jun 2018

Cited by 47 | Viewed by 9027

Abstract

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus [...] Read more.

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8⁺ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials. Full article

(This article belongs to the Special Issue Oncolytic Virotherapy)

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Figure 1

Figure 1
Reovirus has both direct oncolytic effects and induces an inflammatory immune response in breast cancer cells. (A) ED50 of established human and murine breast cancer cell lines infected with serial dilutions of reovirus (RV) multiplicity of infection (MOI) and incubated for 48 h. Cytotoxicity was detected by measuring mitochondrial NADPH dehydrogenase using a (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST) assay. N = 3 per group. (B) EMT6 cells infected with ED50 (7.37 MOI) of UV-irradiated dead reovirus (DV) or live reovirus (LV) for 48 h, taken with a Zeiss Axiovert 200M microscope at 10× zoom. Scale bar = 50 μm. (C) EMT6 cells +/− ED 50 (7.37 MOI) of DV or LV and incubated for 24 h. Chemokine and cytokine levels in supernatants from EMT6 cells were determined by luminex analysis. N = 3 per group. (D) Dendritic cell or (E) Lymphocyte migration in response to cytokine secretion from EMT6 infected or not by RV using of a Transwell® migration assay. N = 4 per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = standard error of the mean (SEM) of three independent experiments. Full article ">Figure 2
Reovirus modulates PD-L1 expression on breast cancer cell lines. Human (A) MDA-MB-468 (B) Hs 578T and murine (C) 4T1 (D) EMT6 breast cancer cell lines were either treated with ED50 RV +/− IFN-γ or DV +/− IFN-γ. Expression of surface PD-L1 was analyzed via surface flow cytometry. N = 3 per group.(E) EMT6 or (F) 4T1 cells were incubated with UV-inactivated supernatant from 4T1 or EMT6 cells, respectively, previously treated with RV or DV for 24 h. PDL-1 expression was analyzed by surface flow cytometry. N = 3 per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = SEM of three independent experiments. Full article ">Figure 3
Reovirus combined with PD-1 inhibition results in decreased tumor burden and improved overall survival in the EMT6 murine model. Balb/C mice were implanted with EMT6 (2 × 105 cells) into the right mammary fat pad and treated with phosphate buffered saline (PBS), anti-PD-1 antibody (200 ug i.p.), RV (5 × 108 PFU i.t.) or a combination of these agents. RV was administered four times (days 6, 9, 12 and 14) following tumor implantation and anti-PD-1 antibody was given six times (days 14. 17, 20, 23, 26 and 29). (A) Tumor size was followed with caliper measurements every three days starting from day 9. PBS N = 15, RV N = 13, PD-1 N = 13, RV + PD-1 N = 14. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by two-way. Error bars = SEM of replicates within each group. (B) Kaplan–Meier survival plot of mice in each treatment group. PBS N = 10, RV N = 8, PD-1 N = 8, RV + PD-1 N = 9. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by log rank test. Error bars = SEM of replicates within each group. Full article ">Figure 4
Reovirus combined with PD-1 inhibition enhances splenic immune stimulatory cells while preventing accumulation of tumor immune suppressor cells. Pooled splenocytes (A–D) and tumor single-cell suspensions (E) from EMT6 tumor–bearing mice treated as per <a href="#cancers-10-00205-f002" class="html-fig">Figure 2</a>A were immunophenotyped by flow cytometry. (A) CD4+ T cells, (B) CD8+ T cells, (C) Effector CD4+ memory T cells, (D) Effector CD8+ memory T cells, (E) T-regulatory cells. N = 5 mice per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = SEM of experimental replicates. Source of cells indicated in parentheses. CD8+ cells were separated from pooled spleens of EMT6 tumor-bearing mice treated as per <a href="#cancers-10-00205-f002" class="html-fig">Figure 2</a>A and stimulated with EMT6 cells. (F) Percentage of EMT6-specific IFN+ cells determined by ELISPOT assay and (G) representative quantification. N = 3 mice per group. *** p ≤ 0.001 by one-way ANOVA. Error bars = SEM of experimental replicates. Full article ">Figure 5
Reovirus combined with PD-1 inhibition significantly enhances IFN-γ, TNF-α and IL-2 production by CD4 and CD8 T cells. Splenocytes were stimulated with Ionomycin for 12 h with Brefeldin A added in the last two hours. Surface and intracellular flow cytometric analysis were performed and stained for markers specific for CD4 T cells (A,C,E) and CD8 T cells (B,D,F). Cytokine secretion for each population was then analyzed [IFN-γ (A/B), TNF-α (C/D) and IL-2 (E/F)]. N = 5 per group. *** p ≤ 0.001, ** p ≤ 0.01 and * p ≤ 0.05 by one-way ANOVA. Error bars = SEM of experimental replicates. Full article ">Figure 6
Survival advantage of combination therapy relies on presence of CD8+ T cells. (A,B) Kaplan–Meier plot demonstrating overall survival (OS )for mice pretreated with depleting CD8a (A) or CD4a (B) antibodies (i.p.) followed by treatment as in <a href="#cancers-10-00205-f003" class="html-fig">Figure 3</a>A,B. ** p ≤ 0.01 and * p ≤ 0.05 by log-rank test. N = 5 mice. Results from <a href="#cancers-10-00205-f003" class="html-fig">Figure 3</a>B included in panel A,B as reference. (C,D) Cohorts of pretreated mice demonstrating cure (Combination: N = 6, RV: N = 3) and a cohort of treatment-naïve mice (N = 5) were challenged with EMT6 (1 × 105 cells) into the opposite (left) mammary fat pad as initial tumor inoculation. (C) Tumor size was followed with caliper measurements every three days starting from day 9. (D) Kaplan–Meier survival plot of mice in each treatment group. ***p ≤ 0.001. Error Bars = SEM of experimental replicates. Full article ">

15 pages, 980 KiB

Open AccessReview

The Role of Immune Checkpoint Inhibitors in Classical Hodgkin Lymphoma

by Nicholas Meti, Khashayar Esfahani and Nathalie A. Johnson

Cancers 2018, 10(6), 204; https://doi.org/10.3390/cancers10060204 - 15 Jun 2018

Cited by 29 | Viewed by 7500

Abstract

Hodgkin Lymphoma (HL) is a unique disease entity both in its pathology and the young patient population that it primarily affects. Although cure rates are high, survivorship can be linked with significant long-term morbidity associated with both chemotherapy and radiotherapy. The most significant [...] Read more.

Hodgkin Lymphoma (HL) is a unique disease entity both in its pathology and the young patient population that it primarily affects. Although cure rates are high, survivorship can be linked with significant long-term morbidity associated with both chemotherapy and radiotherapy. The most significant recent advances have been with the use of the anti-CD30-drug conjugated antibody brentuximab vedotin (BV) and inhibitors of program death 1 (PD-1). HL is genetically wired to up-regulate program death ligand 1 (PD-L1) in >95% of cases, creating a state of so-called “T cell exhaustion”, which can be reversed with immune checkpoint-inhibitor blockade. The overall and complete response rates to PD-1 inhibitors in patients with relapsed or refractory HL are 70% and 20%, respectively, with a long median duration of response of ~16 months. In fact, PD-1 inhibitors can benefit a wide spectrum of relapsed HL patients, including some who have “progressive disease” by strict response criteria. We review the biology of HL, with a focus on the immune micro-environment and mechanisms of immune evasion. We also provide the rationale supporting the use of PD-1 inhibitors in HL and highlight some of the challenges of monitoring disease response in patients treated with this immunotherapy. Full article

(This article belongs to the Special Issue Hodgkin's Lymphoma)

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13 pages, 260 KiB

Open AccessPerspective

Biomarkers for Early Diagnosis and Prognosis of Malignant Pleural Mesothelioma: The Quest Goes on

by Caterina Ledda, Paola Senia and Venerando Rapisarda

Cancers 2018, 10(6), 203; https://doi.org/10.3390/cancers10060203 - 15 Jun 2018

Cited by 40 | Viewed by 10844

Abstract

Malignant pleural mesothelioma (MM) is a highly aggressive tumor characterized by a poor prognosis. Although its carcinogenesis mechanism has not been strictly understood, about 80% of MM can be attributed to occupational and/or environmental exposure to asbestos fibers. The identification of non-invasive molecular [...] Read more.

Malignant pleural mesothelioma (MM) is a highly aggressive tumor characterized by a poor prognosis. Although its carcinogenesis mechanism has not been strictly understood, about 80% of MM can be attributed to occupational and/or environmental exposure to asbestos fibers. The identification of non-invasive molecular markers for an early diagnosis of MM has been the subject of several studies aimed at diagnosing the disease at an early stage. The most studied biomarker is mesothelin, characterized by a good specificity, but it has low sensitivity, especially for non-epithelioid MM. Other protein markers are Fibulin-3 and osteopontin which have not, however, showed a superior diagnostic performance. Recently, interesting results have been reported for the HMGB1 protein in a small but limited series. An increase in channel proteins involved in water transport, aquaporins, have been identified as positive prognostic factors in MM, high levels of expression of aquaporins in tumor cells predict an increase in survival. MicroRNAs and protein panels are among the new indicators of interest. None of the markers available today are sufficiently reliable to be used in the surveillance of subjects exposed to asbestos or in the early detection of MM. Our aim is to give a detailed account of biomarkers available for MM. Full article

(This article belongs to the Special Issue Cancer Biomarkers)

11 pages, 1576 KiB

Open AccessArticle

Loss of Cyclin-Dependent Kinase Inhibitor Alters Oncolytic Adenovirus Replication and Promotes More Efficient Virus Production

by Naseruddin Höti, Tamara Jane Johnson, Wasim H. Chowdhury and Ronald Rodriguez

Cancers 2018, 10(6), 202; https://doi.org/10.3390/cancers10060202 - 15 Jun 2018

Cited by 2 | Viewed by 3709

Abstract

We elucidate the role of p21/Waf-1, a cyclin-dependent kinase inhibitor, on the oncolytic infection and replication cycle of adenovirus by studying both mRNA and adenoviral proteins expression. We found that infection in the absence of p21 causes a significant increase in adenoviral genomes [...] Read more.

We elucidate the role of p21/Waf-1, a cyclin-dependent kinase inhibitor, on the oncolytic infection and replication cycle of adenovirus by studying both mRNA and adenoviral proteins expression. We found that infection in the absence of p21 causes a significant increase in adenoviral genomes and late gene expression. Similarly, the oncolytic adenoviral infected p21^−/− cells have earlier formation of replication foci and robust replication kinetics that were not observed in the wild type p21/Waf-1 intact cells. These findings suggest a culmination that the presence of intact p21 in host cells causes defects in the oncolytic viral life cycle which results in the production of immature and noninfectious particles. Full article

(This article belongs to the Special Issue Oncolytic Virotherapy)

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39 pages, 4806 KiB

Open AccessEditor’s ChoiceReview

Designer Oncolytic Adenovirus: Coming of Age

by Alexander T. Baker, Carmen Aguirre-Hernández, Gunnel Halldén and Alan L. Parker

Cancers 2018, 10(6), 201; https://doi.org/10.3390/cancers10060201 - 14 Jun 2018

Cited by 65 | Viewed by 12878

Abstract

The licensing of talimogene laherparepvec (T-Vec) represented a landmark moment for oncolytic virotherapy, since it provided unequivocal evidence for the long-touted potential of genetically modified replicating viruses as anti-cancer agents. Whilst T-Vec is promising as a locally delivered virotherapy, especially in combination with [...] Read more.

The licensing of talimogene laherparepvec (T-Vec) represented a landmark moment for oncolytic virotherapy, since it provided unequivocal evidence for the long-touted potential of genetically modified replicating viruses as anti-cancer agents. Whilst T-Vec is promising as a locally delivered virotherapy, especially in combination with immune-checkpoint inhibitors, the quest continues for a virus capable of specific tumour cell killing via systemic administration. One candidate is oncolytic adenovirus (Ad); it’s double stranded DNA genome is easily manipulated and a wide range of strategies and technologies have been employed to empower the vector with improved pharmacokinetics and tumour targeting ability. As well characterised clinical and experimental agents, we have detailed knowledge of adenoviruses’ mechanisms of pathogenicity, supported by detailed virological studies and in vivo interactions. In this review we highlight the strides made in the engineering of bespoke adenoviral vectors to specifically infect, replicate within, and destroy tumour cells. We discuss how mutations in genes regulating adenoviral replication after cell entry can be used to restrict replication to the tumour, and summarise how detailed knowledge of viral capsid interactions enable rational modification to eliminate native tropisms, and simultaneously promote active uptake by cancerous tissues. We argue that these designer-viruses, exploiting the viruses natural mechanisms and regulated at every level of replication, represent the ideal platforms for local overexpression of therapeutic transgenes such as immunomodulatory agents. Where T-Vec has paved the way, Ad-based vectors now follow. The era of designer oncolytic virotherapies looks decidedly as though it will soon become a reality. Full article

(This article belongs to the Special Issue Oncolytic Virotherapy)

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Adenovirus Structure. Cartoon view of adenovirus, highlighting the major capsid proteins as labelled (A). Structural view of an adenovirus vertex modelled from CryoEM structure (PDB: 6B1T) showing penton (green) with hexons (dark and light blue) and minor capsid proteins (red) (B). Full article ">Figure 2
Schematic representation of E1A-mediated regulation of the cell cycle. The E1A adenoviral protein promotes S-phase induction by interacting with pRB and p300. Full article ">Figure 3
Coxsackie and Adenovirus Receptor (CAR) interacting residues within the Ad5 fiber knob domain. Known CAR interacting residues are shown as green sticks. The blue and yellow mesh shows the surface of the KO1 and ΔTAYT mutations, respectively. Structure from PDB: 1KNB. Full article ">Figure 4
Membrane cofactor (CD46) interacting residues, and known mutation sites, within the Ad35 fiber knob domain: The key residues which interact with CD46 are shown as green sticks. The yellow surface shows the region in which known mutations which abrogate CD46 interaction occur. In the top right is a detailed view of the 4 loops which interact with CD46, HI (blue), DG (yellow), GH (red), and IJ (green). Structure from PDB: 2QLK. Full article ">Figure 5
DSG2 interacting residues, and known mutation sites, within the Ad3 fiber knob domain: The key residues which interact with DSG2 are shown as green sticks. The yellow surface shows the region in which known mutations which abrogate DSG2 interaction occur. Structure from PDB: 1H7Z. Full article ">Figure 6
GD1a/Sialic Acid interacting residues within the Ad37 fiber knob domain. Key residues forming the GD1a-Ad37Fkn interaction are shown as green sticks, with the GD1a in orange, hydrogen bonds are shown by red dashes. While the interface can occur in three orientations, only one set of interacting residues is shown. The blue dots show the surface of all residues shown to be able to interact with GD1a or support the interaction, seen to create a large apical binding pocket. Structure from PDB: 3N0I. Full article ">Figure 7
Overview of antibody-like proteins specific to Human Epidermal growth factor Receptor 2 (HER2) which have been genetically integrated into Adenovirus. HER2 is composed of 4 domains, Extra Cellular Domain (ECD) I-IV which can be bound by different proteins (A). The Designed Ankyrin Repeat Proteins (DARPIN’s) 9.29 (B) and G3 (C) are seen in orange and red complexing ECD-I and ECD-IV, respectively. ScFv chA21 (D) is seen in purple complexing ECD-1 (this particular ScFv has not previously been integrated to Adenovirus), and affibody ZHER2 (E) is seen in green binding at the ECD-III/IV interface. All molecules are shown to scale, structures from PDB: 4HRL, 4HRN, 3H3B, and 3MZW. Full article ">

15 pages, 1331 KiB

Open AccessArticle

Overcoming Resistance of Human Non-Hodgkin’s Lymphoma to CD19-CAR CTL Therapy by Celecoxib and Histone Deacetylase Inhibitors

by Antoni Xavier Torres-Collado and Ali R. Jazirehi

Cancers 2018, 10(6), 200; https://doi.org/10.3390/cancers10060200 - 14 Jun 2018

Cited by 34 | Viewed by 5882

Abstract

Patients with B-cell non-Hodgkin’s lymphoma (B-NHL) who fail to respond to first-line treatment regimens or develop resistance, exhibit poor prognosis. This signifies the need to develop alternative treatment strategies. CD19-chimeric antigen receptor (CAR) T cell-redirected immunotherapy is an attractive and novel option, which [...] Read more.

Patients with B-cell non-Hodgkin’s lymphoma (B-NHL) who fail to respond to first-line treatment regimens or develop resistance, exhibit poor prognosis. This signifies the need to develop alternative treatment strategies. CD19-chimeric antigen receptor (CAR) T cell-redirected immunotherapy is an attractive and novel option, which has shown encouraging outcomes in phase I clinical trials of relapsed/refractory NHL. However, the underlying mechanisms of, and approaches to overcome, acquired anti-CD19CAR CD8⁺ T cells (CTL)-resistance in NHL remain elusive. CD19CAR transduced primary human CTLs kill CD19⁺ human NHLs in a CD19- and caspase-dependent manner, mainly via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway. To understand the dynamics of the development of resistance, we analyzed several anti-CD19CAR CTL-resistant NHL sublines (R-NHL) derived by serial exposure of sensitive parental lines to excessive numbers of anti-CD19CAR CTLs followed by a limiting dilution analysis. The R-NHLs retained surface CD19 expression and were efficiently recognized by CD19CAR CTLs. However, R-NHLs developed cross-resistance to CD19CAR transduced human primary CTLs and the Jurkat human T cell line, activated Jurkat, and lymphokine activated killer (LAK) cells, suggesting the acquisition of resistance is independent of CD19-loss and might be due to aberrant apoptotic machinery. We hypothesize that the R-NHL refractoriness to CD19CAR CTL killing could be partially rescued by small molecule sensitizers with apoptotic-gene regulatory effects. Chromatin modifiers and Celecoxib partially reversed the resistance of R-NHL cells to the cytotoxic effects of anti-CD19CAR CTLs and rhTRAIL. These in vitro results, though they require further examination, may provide a rational biological basis for combination treatment in the management of CD19CAR CTL-based therapy of NHL. Full article

(This article belongs to the Special Issue CAR-T Cell Therapy-Novel Approaches and Challenges)

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CD19 chimeric antigen receptor (CAR) transduced primary human CD8+ T cells (CTLs) kill CD19+ human non-Hodgkin’s lymphoma (NHL) cell lines. (A) The transduction efficiency of CD19CAR transduced lymphocytes was measured by fluorescence-activated cell sorting (FACS) analysis as detailed in the Materials and Methods section. The results are representative of at least two independent experiments); (B) The sensitivity of various CD19+ NHL lines (Ramos, Raji, Daudi) to CD19CAR CTL killing; (C) CD19CAR CTLs kill NHL cells in a CD19-specific, and caspase- and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated manner. Ramos NHL cells were left either untreated or pretreated with TRAIL, CD19 blocking mAb or zVAD-fmk (1.0 μg/mL, 6 h) and were used in a 51Cr-release assay using CD19CAR CTLs as effectors. BCBL-1 NHL cells were used as a negative control; (D) The specificity of CD19CAR Jurkat killing of NHL. The killing of NHL cells by CD19CAR transduced Jurkat cells (sorted to 100% purity) was significantly reduced by pretreatment of the cells with anti-CD19 mAb (1 μg/mL, 6 h). (E) Killing of the CD19 negative NHL B-cell line, BCBL-1, by various immune effector cells. * p values < 0.05 are considered to be significant. Full article ">Figure 2
Generation of CD19CAR CTL-resistant (R)-NHL sublines. The sensitivity of NHL lines to killing mediated by various immune effector cells. (A) CD19CAR Jurkat (sorted to 100% purity); (B) activated (non-transduced Jurkat T cell line); (C) lymphokine activated killer (LAK) cells. Cross-resistance of CD19CAR CTL-resistant NHL to various immune effector cells; (D) CD19CAR CTL; (E) CD19CAR Jurkat (sorted to 100% purity); (F) activated (non-transduced) Jurkat T cell line (3000 IU/mL IL-2); (G) lymphokine activated killer (LAK) cells. NHL cells (Ramos, Raji, Daudi) were labeled with 51Chromium (1 h, 37 °C), washed 2× with ice-cold PBS and used in standard 51Cr-release assay. p values < 0.05 are considered to be significant. Full article ">Figure 3
Recognition of resistant-NHL sublines by CD19CAR CTLs. (A) Surface expression of CD19 and CD20 on NHL lines: NHL cells (106) were either stained with CD19 and CD20 isotype control or fluorochrome labeled anti-CD19 [FITC]/anti-CD20 [PE] mAbs, as detailed in the Materials and Methods section and subjected to FACS analysis. The results of two independent experiments are presented; (B) Recognition of NHL cells by CD19CAR CTL: 106 tumors were co-incubated overnight with CD19CAR CTLs at a 1:1 E:T ratio. The supernatants were collected and the amount of IFN-γ released was measured using ELISA. The CD19− NHL line, BCBL-1, M202 melanoma line, CD19CAR CTLs, and PBLs were used as controls. Samples were set up in triplicate. The results are presented as means ± SEMs of two independent experiments. Full article ">Figure 4
HDACi (SAHA and LBH589) and celecoxib (Celebrex) reverse NHL resistance to CD19CAR CTL killing. CD19CAR CTL-resistant Ramos cells (106) were left either untreated or pretreated with suberoylanilide hydroxamic acid (SAHA) (1.0 μmol/L), panobinostat (LBH) (0.5 μmol/L), and celecoxib (5.0 μmol/L) for 48 h. The cells were then washed 2× and labeled with 51Chromium (1 h, 37 °C). Thereafter, cells were washed 2× with ice cold PBS and used in a standard 51Cr-release assay using CD19CAR CTLs as effectors. The results are presented as means ± SEMs of duplicate samples. * p values < 0.05 were considered to be significant. Full article ">

18 pages, 10279 KiB

Open AccessReview

TGF-β Sustains Tumor Progression through Biochemical and Mechanical Signal Transduction

by Robert L. Furler, Douglas F. Nixon, Christine A. Brantner, Anastas Popratiloff and Christel H. Uittenbogaart

Cancers 2018, 10(6), 199; https://doi.org/10.3390/cancers10060199 - 14 Jun 2018

Cited by 34 | Viewed by 6360

Abstract

Transforming growth factor β (TGF-β) signaling transduces immunosuppressive biochemical and mechanical signals in the tumor microenvironment. In addition to canonical SMAD transcription factor signaling, TGF-β can promote tumor growth and survival by inhibiting proinflammatory signaling and extracellular matrix (ECM) remodeling. In this article, [...] Read more.

Transforming growth factor β (TGF-β) signaling transduces immunosuppressive biochemical and mechanical signals in the tumor microenvironment. In addition to canonical SMAD transcription factor signaling, TGF-β can promote tumor growth and survival by inhibiting proinflammatory signaling and extracellular matrix (ECM) remodeling. In this article, we review how TGF-β activated kinase 1 (TAK1) activation lies at the intersection of proinflammatory signaling by immune receptors and anti-inflammatory signaling by TGF-β receptors. Additionally, we discuss the role of TGF-β in the mechanobiology of cancer. Understanding how TGF-β dampens proinflammatory responses and induces pro-survival mechanical signals throughout cancer development is critical for designing therapeutics that inhibit tumor progression while bolstering the immune response. Full article

(This article belongs to the Special Issue TGF-Beta Signaling in Cancer)

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T cells and macrophages exhibit immunosuppressive qualities in tumor microenvironments. Despite presence of macrophages (larger egg-like cell in scanning electron microscopy image taken by our group) and T cells (two smaller cells scanning the surface of the macrophage), transforming growth factor β1 (TGF-β1) in the tumor microenvironment inhibited proinflammatory signaling in these leukocytes. Full article ">Figure 2
Transforming growth factor β (TGF-β) dampens anti-tumor proinflammatory signaling in infiltrating leukocytes through TGF-β activated kinase 1 (TAK1), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein kinase (MAPK) modulation: tumor necrosis factor receptor (TNFR)-associated factor (TRAF) and TAK1 activity is regulated by the anti-inflammatory TGF-β receptor (TGF-βR) and proinflammatory interleukin 1 receptor (IL-1R), TNFRs, T cell receptors (TCRs), and B cell receptors (BCRs). TGF-β signaling interferes with TRAF and TAK1 activation to alter NFκB signaling in tumor-associated leukocytes to blunt immune responses during cancer progression. TGF-β modulation of the p38/JNK MAPK pathways and SMAD activity also play a role in inhibiting proinflammatory signals. Full article ">Figure 3
Transforming growth factor β (TGF-β) signaling is intimately linked with the mechanobiology of cells. Dynamic reciprocity is the concept of bidirectional influence of cells and their microenvironment, including adhesions to the extracellular matrix (ECM) and to surrounding cells. Physical linkages between the extracellular microenvironment are created by plasma membrane adhesion receptors, the cytoskeleton, nuclear membrane KASH (Klarsicht, ANC-1, Syne Homology)- and SUN (Sad1p, UNC-84)-domain proteins, and chromatin. KASH- and SUN-domain proteins bind to each other to form linker of nucleoskeleton and cytoskeleton (LINC) complexes that connect the cytoskeleton to the nucleoskeleton. TGF-β signaling plays an important role in this dynamic reciprocity at both the ECM and nuclear levels. Inactive TGF-β binds to latency-associated peptide (LAP) or latent TGF-β binding proteins (LTBPs) in the ECM. ECM degradation or remodeling relieves mechanical tension on the cell down to the nuclear level while simultaneously increasing the bioavailability of active TGF-β. DNA damage can also decrease the structural integrity of this mechanical tension. TGF-β signaling restores this mechanical homeostasis through upregulation of ECM components and DNA repair enzymes. Tumor cells with elevated TGF-β signaling are able to restore mechanical homeostasis despite ongoing ECM remodeling and DNA damage. Full article ">Figure 4
Extracellular matrix (ECM) interactions provide mechanical cues to infiltrating cells. ECM remodeling is required for tumor proliferation, metastasis, angiogenesis, and leukocyte infiltration. The image on the left shows a region with multidirectional bundles of collagen (arrows). The image on the right shows highly organized bundles of parallel collagen fibers found in body (arrow). Dense collagen networks such as these found in capsular regions of lymphoid tissue can be found within the tumor microenvironment. Collagen induced by transforming growth factor β (TGF-β) and other factors provide structural support to cancer cells and a physical barrier to leukocytes. (Transmission electron microscopy images taken by our group.) Full article ">Figure 5
Cell adhesions to other cells and the extracellular matrix (ECM) are mechanically linked to the nucleus. Leukocytes and metastatic tumors often migrate to secondary lymphoid tissues. As seen from the lymphoid tissue images above, cells are intimately connected through several cell–cell adhesions (green diamonds) as they migrate and proliferate in this environment. The four images above show that cell adhesions (green diamonds) are directly linked to the nuclei (red stars) by cytoskeletal elements (black arrows). (Transmission electron microscopy images taken by our group). Full article ">Figure 6
The cytoskeleton can alter nuclear shape. Cell adhesions are directly linked to the nuclear lamina and chromatin via the cytoskeleton and linker of nucleoskeleton and cytoskeleton (LINC) complexes. Forces applied to cellular adhesions have been shown to change nuclear shape and alter gene expression. Deformations at the site of cytoskeletal attachment to the nucleus can be seen in the four images above. As shown in these leukocytes, the fibrillar cytoskeletal elements are connected directly to the nucleus (arrows), primarily at sites of darkened areas of heterochromatin. Microtubules are organized at the centrosome, which contains centrioles. Microtubules are one type of cytoskeletal protein that links adhesion receptors to the nucleus. The bottom right image shows a pair of centrioles and their close proximity to the nuclear envelope (arrow). (Transmission electron microscopy images taken by our group.) Full article ">Figure 7
Parallel organization of extracellular matrix (ECM) fibrils can be used as migration tracts. Transforming growth factor β (TGF-β)-induced fibrosis and ECM remodeling during cancer transmits mechanical information from the tumor microenvironment directly to the nucleus of tumor cells, infiltrating leukocytes, fibroblasts, and endothelial cells. Some metastasizing cancer cells use parallel ECM fibrils, called “tumor-associated collagen signatures” (TACSs), similar to the collagen tracts (arrows) used by the leukocytes shown above migrating through lymphoid tissue. (Transmission electron microscopy images taken by our group.) Full article ">

16 pages, 961 KiB

Open AccessReview

Oncolytic Viruses for Multiple Myeloma Therapy

by Christine M. Calton, Kevin R. Kelly, Faiz Anwer, Jennifer S. Carew and Steffan T. Nawrocki

Cancers 2018, 10(6), 198; https://doi.org/10.3390/cancers10060198 - 14 Jun 2018

Cited by 20 | Viewed by 6827

Abstract

Although recent treatment advances have improved outcomes for patients with multiple myeloma (MM), the disease frequently becomes refractory to current therapies. MM thus remains incurable for most patients and new therapies are urgently needed. Oncolytic viruses are a promising new class of therapeutics [...] Read more.

Although recent treatment advances have improved outcomes for patients with multiple myeloma (MM), the disease frequently becomes refractory to current therapies. MM thus remains incurable for most patients and new therapies are urgently needed. Oncolytic viruses are a promising new class of therapeutics that provide tumor-targeted therapy by specifically infecting and replicating within cancerous cells. Oncolytic therapy yields results from both direct killing of malignant cells and induction of an anti-tumor immune response. In this review, we will describe oncolytic viruses that are being tested for MM therapy with a focus on those agents that have advanced into clinical trials. Full article

(This article belongs to the Special Issue Oncolytic Virotherapy)

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6 pages, 211 KiB

Open AccessCommentary

Antiviral Drugs for EBV

by Joseph S. Pagano, Christopher B. Whitehurst and Graciela Andrei

Cancers 2018, 10(6), 197; https://doi.org/10.3390/cancers10060197 - 13 Jun 2018

Cited by 54 | Viewed by 8416

Abstract

Epstein–Barr virus (EBV) infects up to 95% of the adult human population, with primary infection typically occurring during childhood and usually asymptomatic. However, EBV can cause infectious mononucleosis in approximately 35–50% cases when infection occurs during adolescence and early adulthood. Epstein–Barr virus is [...] Read more.

Epstein–Barr virus (EBV) infects up to 95% of the adult human population, with primary infection typically occurring during childhood and usually asymptomatic. However, EBV can cause infectious mononucleosis in approximately 35–50% cases when infection occurs during adolescence and early adulthood. Epstein–Barr virus is also associated with several B-cell malignancies including Burkitt lymphoma, Hodgkin lymphoma, and post-transplant lymphoproliferative disease. A number of antiviral drugs have proven to be effective inhibitors of EBV replication, yet have resulted in limited success clinically, and none of them has been approved for treatment of EBV infections. Full article

(This article belongs to the Special Issue Epstein–Barr Virus Associated Cancers)

28 pages, 2302 KiB

Open AccessReview

Endogenous Control Mechanisms of FAK and PYK2 and Their Relevance to Cancer Development

by Rayan Naser, Abdullah Aldehaiman, Escarlet Díaz-Galicia and Stefan T. Arold

Cancers 2018, 10(6), 196; https://doi.org/10.3390/cancers10060196 - 11 Jun 2018

Cited by 49 | Viewed by 8154

Abstract

Focal adhesion kinase (FAK) and its close paralogue, proline-rich tyrosine kinase 2 (PYK2), are key regulators of aggressive spreading and metastasis of cancer cells. While targeted small-molecule inhibitors of FAK and PYK2 have been found to have promising antitumor activity, their clinical long-term [...] Read more.

Focal adhesion kinase (FAK) and its close paralogue, proline-rich tyrosine kinase 2 (PYK2), are key regulators of aggressive spreading and metastasis of cancer cells. While targeted small-molecule inhibitors of FAK and PYK2 have been found to have promising antitumor activity, their clinical long-term efficacy may be undermined by the strong capacity of cancer cells to evade anti-kinase drugs. In healthy cells, the expression and/or function of FAK and PYK2 is tightly controlled via modulation of gene expression, competing alternatively spliced forms, non-coding RNAs, and proteins that directly or indirectly affect kinase activation or protein stability. The molecular factors involved in this control are frequently deregulated in cancer cells. Here, we review the endogenous mechanisms controlling FAK and PYK2, and with particular focus on how these mechanisms could inspire or improve anticancer therapies. Full article

(This article belongs to the Special Issue FAK Signaling Pathway in Cancers)

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Figure 1
Schematic domain structure of focal adhesion kinase (FAK) and PYK2. The three folded domains are shown in green, blue and magenta. Interaction motifs, sites of post-translational modification, and examples of binding sites of proteins discussed in the text are shown. The percentages show the sequence identity between corresponding regions of FAK and PYK2. The alternatively transcribed products FRNK and PRNK are schematically represented with respect to FAK and PYK2. Interacting proteins for FAK or PYK2 are shown boxed either above (FAK) or below (PYK2) the schematic structure with arrows pointing at the interacting domain or linker region. For a more complete list of interacting partners, please see [<a href="#B26-cancers-10-00196" class="html-bibr">26</a>]. Full article ">Figure 2
Canonical FAK activation scheme. (A) In the absence of integrin activation, an interaction between the FERM and kinase domain (indicated by a lower-case ‘a’ in a red triangle) inhibits FAK kinase activity. (B) Ligand-mediated recruitment and clustering of FAK at focal adhesions promotes transient dimerization by stabilizing weak FERM:FERM interactions (lower-case ‘b’ in a red triangle) and promoting FERM:FAT binding in trans (lower-case ‘c’ in a red triangle). (C) FAK clustering and self-association allows trans-autophosphorylation of Y397 (red dots) in the FERM-kinase linker. When phosphorylated, Y397 and PR1 form a bidentate binding site for the SH2 and SH3 domains of Src (lower-case ‘d’ in a red triangle). Recruitment-activated Src phosphorylates the activation loop of the FAK kinase domain (lower-case ‘e’ in a red triangle) and other tyrosines on FAK, resulting in an open FAK conformation and full enzymatic activity. Triggered signaling may result in additional FAK modifications (e.g., serine phosphorylation; yellow dots) and may ultimately lead to dephosphorylation and/or displacement of FAK from focal adhesions (back to the closed monomeric inactive conformation of A), or to proteolytic cleavage and degradation (not shown in figure). Full article ">Figure 3
Schematic domain structures (left) and three-dimensional (3D) architectures (right) of protein regulators of FAK:LKB1, PTEN and FIP200 are negative regulators. LKB1 is shown as part of the activating complex formed with MO25α and STRADα (according to PDB accession 2wtk). The presented 3D structure of FIP200 (blue) is highly speculative and based on secondary structure predictions and homology modeling. The stabilizing function of HSP90 is upregulated in cancers. HSP90 is displayed with the kinase-specific adaptor CDC37, maintaining the separated N-terminal (NT) and C-terminal (CT) lobe of a kinase domain (taken from PDB 5fwl). Binding sites and post-transcriptional modifications relevant to their interaction with FAK and PYK2 are indicated. Abbreviations are: (A) LKB1:NTD, N-terminal domain; CTD, C-terminal domain. (B) PTEN:PD, phosphatase domain; PBM, PI(4,5)P2-binding module; PDZ-BD: PDZ binding domain. (C/D) FIP200/HSP90:NT, N-terminal domain; MD: middle domain; CT, C-terminal domain. FAK:KD, kinase domain; NT, N-terminal fragment. Full article ">Figure 4
Overview of selected endogenous mechanisms that influence functions of FAK or PYK2. The ‘Mechanism’ column shows examples of features that affect FAK and PYK2 function, i.e., their local protein concentration, the total amount of correctly folded protein, the degree of tyrosine phosphorylation, and the existence and stability of intramolecular associations, in particular the FERM:kinase autoinhibitory interaction. Selected examples of factors that decrease or increase these mechanisms are shown. For FIP200, the question mark indicates that its influence on intramolecular associations is highly speculative. Full article ">

15 pages, 1147 KiB

Open AccessArticle

Ensuring the Safety and Security of Frozen Lung Cancer Tissue Collections through the Encapsulation of Dried DNA

by Kevin Washetine, Mehdi Kara-Borni, Simon Heeke, Christelle Bonnetaud, Jean-Marc Félix, Lydia Ribeyre, Coraline Bence, Marius Ilié, Olivier Bordone, Marine Pedro, Priscilla Maitre, Virginie Tanga, Emmanuelle Gormally, Pascal Mossuz, Philippe Lorimier, Charles Hugo Marquette, Jérôme Mouroux, Charlotte Cohen, Sandra Lassalle, Elodie Long-Mira, Bruno Clément, Georges Dagher, Véronique Hofman and Paul Hofman Show full author list Hide full author list

Cancers 2018, 10(6), 195; https://doi.org/10.3390/cancers10060195 - 11 Jun 2018

Cited by 5 | Viewed by 3942

Abstract

Collected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the [...] Read more.

Collected specimens for research purposes may or may not be made available depending on their scarcity and/or on the project needs. Their protection against degradation or in the event of an incident is pivotal. Duplication and storage on a different site is the best way to assure their sustainability. The conservation of samples at room temperature (RT) by duplication can facilitate their protection. We describe a security system for the collection of non-small cell lung cancers (NSCLC) stored in the biobank of the Nice Hospital Center, France, by duplication and conservation of lyophilized (dried), encapsulated DNA kept at RT. Therefore, three frozen tissue collections from non-smoking, early stage and sarcomatoid carcinoma NSCLC patients were selected for this study. DNA was extracted, lyophilized and encapsulated at RT under anoxic conditions using the DNAshell technology. In total, 1974 samples from 987 patients were encapsulated. Six and two capsules from each sample were stored in the biobanks of the Nice and Grenoble (France) Hospitals, respectively. In conclusion, DNA maintained at RT allows for the conservation, duplication and durability of collections of interest stored in biobanks. This is a low-cost and safe technology that requires a limited amount of space and has a low environmental impact. Full article

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21 pages, 950 KiB

Open AccessEditor’s ChoiceReview

TGF-β in T Cell Biology: Implications for Cancer Immunotherapy

by Amina Dahmani and Jean-Sébastien Delisle

Cancers 2018, 10(6), 194; https://doi.org/10.3390/cancers10060194 - 11 Jun 2018

Cited by 134 | Viewed by 13084

Abstract

Transforming Growth Factor beta (TGF-β) is a pleiotropic cytokine produced in large amounts within cancer microenvironments that will ultimately promote neoplastic progression, notably by suppressing the host’s T-cell immunosurveillance. This effect is mostly due to the well-known inhibitory effect of TGF-β on T [...] Read more.

Transforming Growth Factor beta (TGF-β) is a pleiotropic cytokine produced in large amounts within cancer microenvironments that will ultimately promote neoplastic progression, notably by suppressing the host’s T-cell immunosurveillance. This effect is mostly due to the well-known inhibitory effect of TGF-β on T cell proliferation, activation, and effector functions. Moreover, TGF-β subverts T cell immunity by favoring regulatory T-cell differentiation, further reinforcing immunosuppression within tumor microenvironments. These findings stimulated the development of many strategies to block TGF-β or its signaling pathways, either as monotherapy or in combination with other therapies, to restore anti-cancer immunity. Paradoxically, recent studies provided evidence that TGF-β can also promote differentiation of certain inflammatory populations of T cells, such as Th17, Th9, and resident-memory T cells (Trm), which have been associated with improved tumor control in several models. Here, we review current advances in our understanding of the many roles of TGF-β in T cell biology in the context of tumor immunity and discuss the possibility to manipulate TGF-β signaling to improve cancer immunotherapy. Full article

(This article belongs to the Special Issue TGF-Beta Signaling in Cancer)

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14 pages, 402 KiB

Open AccessFeature PaperReview

Emerging Therapies and Future Directions in Targeting the Tumor Stroma and Immune System in the Treatment of Pancreatic Adenocarcinoma

by Daniel H. Ahn, Ramesh K. Ramanathan and Tanios Bekaii-Saab

Cancers 2018, 10(6), 193; https://doi.org/10.3390/cancers10060193 - 11 Jun 2018

Cited by 16 | Viewed by 4802

Abstract

Pancreatic adenocarcinoma is typically refractory to conventional treatments and associated with poor prognosis. While therapeutic advances over the past several years have improved patient outcomes, the observed benefits have been modest at best, highlighting the need for continued development of alternate treatment strategies. [...] Read more.

Pancreatic adenocarcinoma is typically refractory to conventional treatments and associated with poor prognosis. While therapeutic advances over the past several years have improved patient outcomes, the observed benefits have been modest at best, highlighting the need for continued development of alternate treatment strategies. The tumor microenvironment has been identified as being integral to oncogenesis through its direct effect on cellular pathway communication, immune inhibition, and promoting chemo-resistance. A more in depth understanding of the biology of the disease, in addition with our ability to develop more effective novel therapies have led to ongoing studies that are investigating several promising treatment options in this disease. Herein, we highlight and review the therapeutic landscape in pancreatic adenocarcinoma. Full article

(This article belongs to the Special Issue Latest Development in Pancreatic Cancer)

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10 pages, 2353 KiB

Open AccessFeature PaperArticle

Recombinant TSR1 of ADAMTS5 Suppresses Melanoma Growth in Mice via an Anti-angiogenic Mechanism

by Bhuvanasundar Renganathan, Vinoth Durairaj, Dogan Can Kirman, Paa Kow A. Esubonteng, Swee Kim Ang and Ruowen Ge

Cancers 2018, 10(6), 192; https://doi.org/10.3390/cancers10060192 - 11 Jun 2018

Cited by 10 | Viewed by 4510

Abstract

Inhibiting tumor angiogenesis is a well-established approach for anticancer therapeutic development. A Disintegrin-like and Metalloproteinase with ThromboSpondin Motifs 5 (ADAMTS5) is a secreted matrix metalloproteinase in the ADAMTS family that also functions as an anti-angiogenic/anti-tumorigenic molecule. Its anti-angiogenic/anti-tumorigenic function is independent from its [...] Read more.

Inhibiting tumor angiogenesis is a well-established approach for anticancer therapeutic development. A Disintegrin-like and Metalloproteinase with ThromboSpondin Motifs 5 (ADAMTS5) is a secreted matrix metalloproteinase in the ADAMTS family that also functions as an anti-angiogenic/anti-tumorigenic molecule. Its anti-angiogenic/anti-tumorigenic function is independent from its proteinase activity, but requires its first thrombospondin type 1 repeat (TSR1). However, it is not known if recombinant TSR1 (rTSR1) can function as an anticancer therapeutic. In this report, we expressed and purified a 75-residue recombinant TSR1 polypeptide from E. coli and investigated its ability to function as an anticancer therapeutic in mice. We demonstrate that rTSR1 is present in the blood circulation as well as in the tumor tissue at 15 min post intraperitoneal injection. Intraperitoneal delivery of rTSR1 potently suppressed subcutaneous B16F10 melanoma growth as a single agent, accompanied by diminished tumor angiogenesis, increased apoptosis, and reduced cell proliferation in the tumor tissue. Consistently, rTSR1 dose-dependently induced the apoptosis of cultured human umbilical vein endothelial cells (HUVECs) in a caspase-dependent manner. This work indicates that rTSR1 of ADAMTS5 can function as a potent anticancer therapy in mice. It thus has the potential to be further developed into an anticancer drug. Full article

(This article belongs to the Special Issue Tumor Angiogenesis: An Update)

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Recombinant type 1 repeat domain (rTSR1) induces caspase-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). (A) SDS-PAGE and immunoblot analysis of purified rTSR1 showed a band at ~10 kDa; (B) rTSR1 dose-dependently induced apoptosis in HUVECs in the presence of VEGF. Data shown are apoptosis observed at 24 h post-1000 nM rTSR1 treatment; (C) Pan-caspase inhibitor (Z-VAD-FMK) significantly reduced the 1000 nM rTSR1-induced apoptosis at 24 h post treatment. Data represent the mean of triplicates ± SD. Statistical analysis performed by one-way ANOVA. * p < 0.05, ** p < 0.01, **** p < 0.0001. Full article ">Figure 2
Systemically delivered rTSR1 suppressed B16F10 tumor growth in mice. (A) Tumor growth curve from day 9 to 15 post tumor cell injection (n = 5). Data represents mean ± SD. The rTSR1 was systemically delivered through intra-peritoneal (IP) injection. Statistical analysis performed by Student’s t test. * p < 0.05; (B) Tumor weight at the end of experiment, control and rTSR1 treated group, n = 5 for each group. The graph represents the mean ± SD of the tumor weight. Statistical analysis performed by Student’s t test. * p < 0.05; (C) Dissected tumor nodules from control and rTSR1 treated groups—scale bar: 1 cm; (D) rTSR1-treated mice showed a reduced peri-tumor vascular network compared to tumors of similar sizes in the control group—scale bar: 0.5 cm. Arrows indicate the blood vessels surrounding the tumor. Full article ">Figure 3
The rTSR1 suppressed angiogenesis and cell proliferation but induced apoptosis in tumors. Tumor paraffin sections were probed for microvessel density, tumor cell proliferation, and apoptosis through immunofluorescence staining, using (A) anti-endomucin, (B) TUNEL staining, and (C) anti-PCNA (Proliferating cell nuclear antigen) respectively. The corresponding quantification of the staining is presented in the bar graph on the right. The percentage of proliferating cells/field is the ratio of the nuclear PCNA-positive cells to the total number of nuclei in the field. Scale bar: 200 μm. Corresponding quantifications are presented in the bar graph on the right. Data represent the mean ± SD of four fields per section, four sections per tumor, and two tumors per group. Statistical analysis was performed by Student’s t test. ** p < 0.01. Full article ">Figure 4
The rTSR1 suppressed tumor endothelial cell proliferation. Representative microscopic images show decreased proliferating endothelial cells in an rTSR1-treated tumor compared to the control. Green indicates the endothelial cells (endomucin-positive), and red indicates the nuclear PCNA-positive proliferating cells. DAPI (4′,6-diamidino-2-phenylindole) was used as the nuclear counter stain. The arrow heads indicate the nuclear PCNA-positive tumor endothelial cells. Scale bar: 30 μm. Full article ">Figure 5
The rTSR1 induced apoptosis in tumor endothelial cells. Representative microscopic images show the increased apoptotic endothelial cells in an rTSR1-treated tumor compared to the control. Red indicates the endothelial cells (endomucin-positive) and green indicates the cleaved caspase-3-positive cells. DAPI was used as the nuclear counter stain. The arrow heads indicates the cleaved caspase-3-positive endothelial cells. Scale bar: 20 μm. Full article ">Figure 6
IP-injected rTSR1 reached the tumor. Immunoblot of serum and tumor lysate displayed band at ~10 kDa, corresponding to the molecular weight of rTSR1. The band is observed both in serum (A) and with the whole tumor lysate (B) from mice that received rTSR1 only. The rTSR1 input is used as the positive control. M1 to M3 refer to mouse 1 to mouse 3. Full article ">

10 pages, 664 KiB

Open AccessCommentary

WIP-YAP/TAZ as A New Pro-Oncogenic Pathway in Glioma

by Sergio Rivas, Inés M. Antón and Francisco Wandosell

Cancers 2018, 10(6), 191; https://doi.org/10.3390/cancers10060191 - 9 Jun 2018

Cited by 18 | Viewed by 5652

Abstract

Wild-type p53 (wtp53) is described as a tumour suppressor gene, and mutations in p53 occur in many human cancers. Indeed, in high-grade malignant glioma, numerous molecular genetics studies have established central roles of RTK-PI3K-PTEN and ARF-MDM2-p53 INK4a-RB pathways in promoting oncogenic capacity. Deregulation [...] Read more.

Wild-type p53 (wtp53) is described as a tumour suppressor gene, and mutations in p53 occur in many human cancers. Indeed, in high-grade malignant glioma, numerous molecular genetics studies have established central roles of RTK-PI3K-PTEN and ARF-MDM2-p53 INK4a-RB pathways in promoting oncogenic capacity. Deregulation of these signalling pathways, among others, drives changes in the glial/stem cell state and environment that permit autonomous growth. The initially transformed cell may undergo subsequent modifications, acquiring a more complete tumour-initiating phenotype responsible for disease advancement to stages that are more aggressive. We recently established that the oncogenic activity of mutant p53 (mtp53) is driven by the actin cytoskeleton-associated protein WIP (WASP-interacting protein), correlated with tumour growth, and more importantly that both proteins are responsible for the tumour-initiating cell phenotype. We reported that WIP knockdown in mtp53-expressing glioblastoma greatly reduced proliferation and growth capacity of cancer stem cell (CSC)-like cells and decreased CSC-like markers, such as hyaluronic acid receptor (CD44), prominin-1 (CD133), yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). We thus propose a new CSC signalling pathway downstream of mtp53 in which Akt regulates WIP and controls YAP/TAZ stability. WIP drives a mechanism that stimulates growth signals, promoting YAP/TAZ and β-catenin stability in a Hippo-independent fashion, which allows cells to coordinate processes such as proliferation, stemness and invasiveness, which are key factors in cancer progression. Based on this multistep tumourigenic model, it is tantalizing to propose that WIP inhibitors may be applied as an effective anti-cancer therapy. Full article

(This article belongs to the Special Issue Hippo Pathway in Cancer, towards Realization of the Hippo-Targeted Therapy)

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14 pages, 39779 KiB

Open AccessArticle

Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation

by Koji Yamasaki, Shoichiro Mukai, Satoru Sugie, Takahiro Nagai, Kozue Nakahara, Toyoharu Kamibeppu, Hiromasa Sakamoto, Noboru Shibasaki, Naoki Terada, Yoshinobu Toda, Hiroaki Kataoka and Toshiyuki Kamoto

Cancers 2018, 10(6), 190; https://doi.org/10.3390/cancers10060190 - 8 Jun 2018

Cited by 5 | Viewed by 4138

Abstract

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone [...] Read more.

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system. Full article

(This article belongs to the Special Issue Targeting Bone Metastasis in Cancer)

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RT-qPCR analyses of MET, a c-met proto-oncogene product and receptor for hepatocyte growth factor (HGF), HGF activator inhibitor-1 (HAI-1), and HAI-2 and matriptase in six renal cell carcinoma cell lines. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the internal control. Full article ">Figure 2
Bioluminescent imaging of the mouse model. After intra-cardiac injection (A), six weeks from injection (B). Apparent bone metastasis is confirmed (B), red circle. Extracted specimens were used for RT-qPCR analyses. mRNA expression of MET, HAI-1, HAI-2, HGF and matriptase in bone metastasis and subcutaneous implantation of 786-O-Luc2 cells (C). Full article ">Figure 2 Cont.
Bioluminescent imaging of the mouse model. After intra-cardiac injection (A), six weeks from injection (B). Apparent bone metastasis is confirmed (B), red circle. Extracted specimens were used for RT-qPCR analyses. mRNA expression of MET, HAI-1, HAI-2, HGF and matriptase in bone metastasis and subcutaneous implantation of 786-O-Luc2 cells (C). Full article ">Figure 3
Pathological and immunohisitochemical appearance of bone metastasis and subcutaneous implantation of 786-O-Luc2 cells using serial tissue sections. (A) Bone metastasis (bone), stained with hematoxylin and eosin (H&E); (B) subcutaneous implantation (SC), stained with H&E; (C) bone, phosphorylation of MET (p-MET) immunostaining; (D) SC, p-MET immunostaining; (E) bone, total-MET (t-MET) immunostaining; (F) SC, t-MET immunostaining. Scale bars = 100 μm. Full article ">Figure 4
(A) Expression of HAI-2 was confirmed by RT-qPCR analyses in HAI-2-knockdown 786-O-Luc2 (786-O-L2898) with and without 0.5 μg/mL of doxycycline (DOC). Effect of HAI-2-knockdown on expression of MET, HAI-1 and matriptase was also verified; (B) Expression of HAI-2, MET, HAI-1 and matriptase were confirmed in HAI-2-engineered expressed 786-O-Luc2 (786-O-L2853) cells with and without 0.5 μg/mL of doxycycline. Full article ">Figure 5
Effect of HAI-2 knockdown or engineered expression on motility and invasiveness of 786-O cells. Cell motility was evaluated by wound healing assay (A), and invasion assay (B) through Matigel with and without endogenous pro-HGF. Values are means ± standard deviation of triplicate experiments. * p < 0.05, Mann–Whitney U test. Full article ">

22 pages, 1504 KiB

Open AccessReview

The Roles of p53 in Mitochondrial Dynamics and Cancer Metabolism: The Pendulum between Survival and Death in Breast Cancer?

by David E. Moulder, Diana Hatoum, Enoch Tay, Yiguang Lin and Eileen M. McGowan

Cancers 2018, 10(6), 189; https://doi.org/10.3390/cancers10060189 - 8 Jun 2018

Cited by 47 | Viewed by 15314

Abstract

Cancer research has been heavily geared towards genomic events in the development and progression of cancer. In contrast, metabolic regulation, such as aberrant metabolism in cancer, is poorly understood. Alteration in cellular metabolism was once regarded simply as a consequence of cancer rather [...] Read more.

Cancer research has been heavily geared towards genomic events in the development and progression of cancer. In contrast, metabolic regulation, such as aberrant metabolism in cancer, is poorly understood. Alteration in cellular metabolism was once regarded simply as a consequence of cancer rather than as playing a primary role in cancer promotion and maintenance. Resurgence of cancer metabolism research has identified critical metabolic reprogramming events within biosynthetic and bioenergetic pathways needed to fulfill the requirements of cancer cell growth and maintenance. The tumor suppressor protein p53 is emerging as a key regulator of metabolic processes and metabolic reprogramming in cancer cells—balancing the pendulum between cell death and survival. This review provides an overview of the classical and emerging non-classical tumor suppressor roles of p53 in regulating mitochondrial dynamics: mitochondrial engagement in cell death processes in the prevention of cancer. On the other hand, we discuss p53 as a key metabolic switch in cellular function and survival. The focus is then on the conceivable roles of p53 in breast cancer metabolism. Understanding the metabolic functions of p53 within breast cancer metabolism will, in due course, reveal critical metabolic hotspots that cancers advantageously re-engineer for sustenance. Illustration of these events will pave the way for finding novel therapeutics that target cancer metabolism and serve to overcome the breast cancer burden. Full article

(This article belongs to the Special Issue p53 Signaling in Cancers)

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16 pages, 424 KiB

Open AccessEditor’s ChoiceReview

Gain-of-Function (GOF) Mutant p53 as Actionable Therapeutic Target

by Ramona Schulz-Heddergott and Ute M. Moll

Cancers 2018, 10(6), 188; https://doi.org/10.3390/cancers10060188 - 7 Jun 2018

Cited by 84 | Viewed by 8731

Abstract

p53 missense mutant alleles are present in nearly 40% of all human tumors. Such mutated alleles generate aberrant proteins that not only lose their tumor-suppressive functions but also frequently act as driver oncogenes, which promote malignant progression, invasion, metastasis, and chemoresistance, leading to [...] Read more.

p53 missense mutant alleles are present in nearly 40% of all human tumors. Such mutated alleles generate aberrant proteins that not only lose their tumor-suppressive functions but also frequently act as driver oncogenes, which promote malignant progression, invasion, metastasis, and chemoresistance, leading to reduced survival in patients and mice. Notably, these oncogenic gain-of-function (GOF) missense mutant p53 proteins (mutp53) are constitutively and tumor-specific stabilised. This stabilisation is one key pre-requisite for their GOF and is largely due to mutp53 protection from the E3 ubiquitin ligases Mdm2 and CHIP by the HSP90/HDAC6 chaperone machinery. Recent mouse models provide convincing evidence that tumors with highly stabilized GOF mutp53 proteins depend on them for growth, maintenance, and metastasis, thus creating exploitable tumor-specific vulnerabilities that markedly increase lifespan if intercepted. This identifies mutp53 as a promising cancer-specific drug target. This review discusses direct mutp53 protein-targeting drug strategies that are currently being developed at various preclinical levels. Full article

(This article belongs to the Special Issue p53 Signaling in Cancers)

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20 pages, 640 KiB

Open AccessReview

Evolving Treatment Strategies for Elderly Leukemia Patients with IDH Mutations

by Michael J. Buege, Adam J. DiPippo and Courtney D. DiNardo

Cancers 2018, 10(6), 187; https://doi.org/10.3390/cancers10060187 - 6 Jun 2018

Cited by 29 | Viewed by 4861

Abstract

Acute myeloid leukemia (AML) is a debilitating and life-threatening condition, especially for elderly patients who account for over 50% of diagnoses. For over four decades, standard induction therapy with intensive cytotoxic chemotherapy for AML had remained unchanged. However, for most patients, standard therapy [...] Read more.

Acute myeloid leukemia (AML) is a debilitating and life-threatening condition, especially for elderly patients who account for over 50% of diagnoses. For over four decades, standard induction therapy with intensive cytotoxic chemotherapy for AML had remained unchanged. However, for most patients, standard therapy continues to have its shortcomings, especially for elderly patients who may not be able to tolerate the complications from intensive cytotoxic chemotherapy. New research into the development of targeted and alternative therapies has led to a new era in AML therapy. For the nearly 20% of diagnoses harboring a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2), potential treatment options have undergone a paradigm shift away from intensive cytotoxic chemotherapy and towards targeted therapy alone or in combination with lower intensity chemotherapy. The first FDA approved IDH2 inhibitor was enasidenib in 2017. In addition, IDH1 inhibitors are in ongoing clinical studies, and the oral BCL-2 inhibitor venetoclax shows preliminary efficacy in this subset of patients. These new tools aim to improve outcomes and change the treatment paradigm for elderly patients with IDH mutant AML. However, the challenge of how to best incorporate these agents into standard practice remains. Full article

(This article belongs to the Special Issue Treatment of Older Adults with Acute Myeloid Leukemia)

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13 pages, 2494 KiB

Open AccessArticle

Regulation of Constitutive Interferon-Stimulated Genes (Isgs) in Tumor Cells Contributes to Enhanced Antitumor Response of Newcastle Disease Virus-Infected Tumor Vaccines

by Mai Takamura-Ishii, Takaaki Nakaya and Katsuro Hagiwara

Cancers 2018, 10(6), 186; https://doi.org/10.3390/cancers10060186 - 6 Jun 2018

Cited by 5 | Viewed by 4585

Abstract

Newcastle disease virus (NDV) is an oncolytic virus. As immunogenicity of tumor cells is enhanced by NDV infection, recombinant NDV-infected tumor vaccines (rNDV-TV) are effective methods for inducing specific immunity. However, several tumor cells resist NDV infection, and tumor specific immunity is not [...] Read more.

Newcastle disease virus (NDV) is an oncolytic virus. As immunogenicity of tumor cells is enhanced by NDV infection, recombinant NDV-infected tumor vaccines (rNDV-TV) are effective methods for inducing specific immunity. However, several tumor cells resist NDV infection, and tumor specific immunity is not sufficiently induced by rNDV-TV. Therefore, we clarified the factor contributing to the suppression of NDV infection and attempted to improve rNDV-TV. Initially we investigated the correlation between the NDV infection rate and interferon-related gene expression in six murine tumor cell lines. A significant negative correlation was observed between the constitutive gene expression of Interferon-stimulated genes (ISGs) and NDV infectivity. The NDV infection rate was examined in each tumor cell treated with the Janus kinase (JAK) inhibitor ruxolitinib (Rux). Furthermore, we evaluated the Th1 response induction by Rux-treated rNDV-TV (rNDV-TV-Rux). In Rux-treated tumor cells, Oasl2 gene expression was significantly decreased and viral infectivity was increased. In immunized mice, the number of CD8⁺ cells, and those expressing the IFN-γ gene, were significantly increased as compared with Rux-untreated rNDV-TV. The infectivity of the virus was dependent on the degree of ISGs expression in tumor cells. To remedy for this problem, rNDV-TV-Rux was expected to have a Th1 immune response. Full article

(This article belongs to the Special Issue Oncolytic Virotherapy)

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Full article ">Figure 1
Newcastle disease virus (NDV)-infected tumor vaccines (NDV-TV) model and induction of antitumor response by rNDV-TV. (a) The schematic of NDV-TV model was shown. The tumor cells were infected with rNDV in vitro, irradiated by ultraviolet for inactivation of rNDV and tumor cells. Then rNDV-TV was administered to mice as immunogen. rNDV-TV (n = 3), UV-TV (ultraviolet irradiated tumor vaccine) (n = 3), or each medium (n = 2) were administered to mice and splenic mononuclear cells (SMCs) were co-cultured with UV-irradiated tumor cells for 5 days. After cytotoxic T cell (CTL) induction, SMCs were harvested and co-cultured with target tumor cells for 24 h. The cytotoxicity was measured using quantifying lactate dehydrogenase (LDH) in the supernatant (* p < 0.05, ** p < 0.01). rNDV-TV and UV-TV were prepared using (b) B16 and (c) SCC VII. Medium was administered to the control mice. Full article ">Figure 2
Recombinant NDV (rNDV) infection rate in murine tumor cell lines. Murine tumor cell lines were infected with rNDV (MOI of 2). The infection rate was calculated from the green fluorescent protein (GFP) expressing cell ratio in fluorescent microscopic observation. Full article ">Figure 3
rNDV infection rate in tumor cells after ruxolitinib treatment. (a) Tumor cells were infected with rNDV (MOI of 2) after ruxolitinib (0~1.0 μg/mL) treatment for 20 h. The infection rate was calculated from the GFP expressing cell ratio observed via fluorescent microscopy (* p < 0.05, ** p < 0.01). (b) GFP expressed in (i) B16, (ii) WEHI164, (iii) 3LL, (iv) SCCVII, (v) MBT-2, and (vi) BALB-MC infected with rNDV after ruxolitinib (left: 0 μg/mL, right: 1.0 μg/mL) treatment (×100 magnification). Full article ">Figure 4
ISGs expression after ruxolitinib. Tumor cells were cultured in medium with (ruxolitinib) or without (mock) ruxolitinib for 20 h. Then, (a, b) Oasl2, (c) Oas1b, and (d) Mx1 gene expression was quantified by qPCR. Gene expression was normalized with GAPDH (* p < 0.05, ** p < 0.01). Full article ">Figure 5
Lymphocyte subsets of SMCs after vaccination. Medium (control, n = 5), MBT-2 using rNDV-TV (MBT-NDV, n = 5), or rNDV-TV-Rux (MBT-NDV-Rux, n = 5) were administered to the mice. Lymphocyte subsets (a) ratio and (b) numbers of SMCs were analyzed by flow cytometry after the last vaccination (* p < 0.05, ** p < 0.01). Full article ">Figure 6
IFN-γ gene expression in MBT-2 stimulated SMCs. SMCs from immunized mice were co-cultured with MBT-2 for 4 h. Then, IFN-γ gene expression of the SMCs were analyzed by qPCR. The gene expression was normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the comparison of gene expression upon stimulation against unstimulated was calculated (** p < 0.01, each group n = 5). Full article ">

14 pages, 1308 KiB

Open AccessFeature PaperEditor’s ChoiceReview

Cutting to the Chase: How Matrix Metalloproteinase-2 Activity Controls Breast-Cancer-to-Bone Metastasis

by Marilena Tauro and Conor C. Lynch

Cancers 2018, 10(6), 185; https://doi.org/10.3390/cancers10060185 - 5 Jun 2018

Cited by 54 | Viewed by 7800

Abstract

Bone metastatic breast cancer is currently incurable and will be evident in more than 70% of patients that succumb to the disease. Understanding the factors that contribute to the progression and metastasis of breast cancer can reveal therapeutic opportunities. Matrix metalloproteinases (MMPs) are [...] Read more.

Bone metastatic breast cancer is currently incurable and will be evident in more than 70% of patients that succumb to the disease. Understanding the factors that contribute to the progression and metastasis of breast cancer can reveal therapeutic opportunities. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes whose role in cancer has been widely documented. They are capable of contributing to every step of the metastatic cascade, but enthusiasm for the use of MMP inhibition as a therapeutic approach has been dampened by the disappointing results of clinical trials conducted more than 20 years ago. Since the trials, our knowledge of MMP biology has expanded greatly. Combined with advances in the selective targeting of individual MMPs and the specific delivery of therapeutics to the tumor microenvironment, we may be on the verge of finally realizing the promise of MMP inhibition as a treatment strategy. Here, as a case in point, we focus specifically on MMP-2 as an example to show how it can contribute to each stage of breast-cancer-to-bone metastasis and also discuss novel approaches for the selective targeting of MMP-2 in the setting of the bone-cancer microenvironment. Full article

(This article belongs to the Special Issue Targeting Bone Metastasis in Cancer)

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17 pages, 4041 KiB

Open AccessFeature PaperArticle

RIPK2: New Elements in Modulating Inflammatory Breast Cancer Pathogenesis

by Alaa Zare, Alexandra Petrova, Mehdi Agoumi, Heather Armstrong, Gilbert Bigras, Katia Tonkin, Eytan Wine and Shairaz Baksh

Cancers 2018, 10(6), 184; https://doi.org/10.3390/cancers10060184 - 5 Jun 2018

Cited by 25 | Viewed by 6082

Abstract

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that is associated with significantly high mortality. In spite of advances in IBC diagnoses, the prognosis is still poor compared to non-IBC. Due to the aggressive nature of the disease, [...] Read more.

Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that is associated with significantly high mortality. In spite of advances in IBC diagnoses, the prognosis is still poor compared to non-IBC. Due to the aggressive nature of the disease, we hypothesize that elevated levels of inflammatory mediators may drive tumorigenesis and metastasis in IBC patients. Utilizing IBC cell models and patient tumor samples, we can detect elevated NF-κB activity and hyperactivation of non-canonical drivers of NF-κB (nuclear factor kappaB)-directed inflammation such as tyrosine phosphorylated receptor-interacting protein kinase 2 (pY RIPK2), when compared to non-IBC cells or patients. Interestingly, elevated RIPK2 activity levels were present in a majority of pre-chemotherapy samples from IBC patients at the time of diagnosis to suggest that patients at diagnosis had molecular activation of NF-κB via RIPK2, a phenomenon we define as “molecular inflammation”. Surprisingly, chemotherapy did cause a significant increase in RIPK2 activity and thus molecular inflammation suggesting that chemotherapy does not resolve the molecular activation of NF-κB via RIPK2. This would impact on the metastatic potential of IBC cells. Indeed, we can demonstrate that RIPK2 activity correlated with advanced tumor, metastasis, and group stage as well as body mass index (BMI) to indicate that RIPK2 might be a useful prognostic marker for IBC and advanced stage breast cancer. Full article

(This article belongs to the Special Issue Inflammation and Cancer)

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Figure 1
NF-κB (nuclear factor kappaB) activity in inflammatory breast cancer (IBC) cell lines. Equal concentrations of total protein from nuclear or cytoplasmic extracts were loaded into a gel and immunoblotted (IB) with the phospho-NF-κB p65 antibody that recognizes the p65 subunit phosphorylated at S536. PCNA (Proliferating cell nuclear antigen) expression is used as a control for nuclear fraction, and Actin is used as a control for cytoplasmic fraction. Signal was developed using enhanced chemiluminescence (ECL). Data shown are representative of the results of at least three independent experiments. Full article ">Figure 2
RIPK2 is hyperactive in IBC cell lines: (a) equal concentrations of samples were loaded and immunoblotted (IB) with an antibody to the active form of RIPK2 using a RIPK2 phospho (p) –Serine (S) 176, RIPK2 phospho (p) -tyrosine (Y) 474 or an antibody that recognizes total RIPK2. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) expression is used as a loading control for whole cell lysate. Signal was developed using enhanced chemiluminescence (ECL). * p-value for the difference between MCF 10A and KPL4, SUM149, and MDA-IBC-3 is 0.01, 0.02, and 0.004, respectively. ** p-value for the difference between MCF7 and KPL4, SUM149, and MDA-IBC-3 are 0.02, 0.03, and 0.001; (b) luminescent ADP-Glo in vitro RIPK2 kinase assay in breast cancer cell lines. RIPK2 was immunoprecipitated and kinase activity was then measured by quantifying luminescence (RLU) that correlates to the percentage of ADP produced during the enzymatic reaction as per manufacture instructions. * p-value for the difference between MCF 10A and KPL4, SUM149, and MDA-IBC-3 are 0.004, 0.09, and 0.04, respectively. ** p-value for the difference between MCF7 and KPL4, SUM149, and MDA-IBC-3 is 0.01, 0.03, and 0.02, respectively; and (c) radioactive in vitro kinase assay in different breast cancer cell lines (cut from the same gel). RIPK2 was immunoprecipitated and kinase activity was then determined using radioactively labeled ATP. The autophosphorylation site of Y474 of RIPK2 is visualized using standard autoradiography. All the data shown are representative of the results of two to four independent experiments. KPL4 revealed similar constitutively active level of RIPK2 [<a href="#B43-cancers-10-00184" class="html-bibr">43</a>]. Full article ">Figure 3
Immunohistochemical staining of normal non-neoplastic breast: (a) luminal A; (b) luminal B; (c) HER2 overexpressed; (d) triple negative breast cancer (TNBC); (e) and IBC; (f) using RIPK2 phospho-Y474 antibody). Breast tissue was stained and visualized using horseradish peroxidase-conjugated secondary antibody and 3, 3′ diaminobenzidine (DAB; brown), red scale bar: 50 µm, black scale bar: 20 µm. DAB staining of luminal A (n = 7), luminal B (n = 8), HER2 overexpressed (n = 7), TNBC (n = 10) and IBC (n = 18). Tissue was quantified using the ImageJ platform permitting integrated optical density assessment of regions of interested in each slide. ImageJ analyzed images were then normalized to normal breast tissue (n = 17) imaged in a similar manner; and (g) the plot represents the fold change in RIPK2 phospho-Y474 expression in tumor tissue relative to normal non-neoplastic breast tissues. p-value is calculated against normal breast tissue. All breast cancer tissues were isolated from patients after neoadjuvant chemotherapy treatment. A description of our patient population is documented in <a href="#cancers-10-00184-t001" class="html-table">Table 1</a> (in the Materials and Methods <a href="#sec4dot3-cancers-10-00184" class="html-sec">Section 4.3</a>). Full article ">Figure 4
Immunohistochemical staining using the RIPK2 phospho-Y474 antibody in IBC breast tissue pre- and post-chemotherapy as indicated. Red scale bar: 50 µm. DAB staining was quantified using ImageJ software and normalized to normal non-neoplastic breast tissue. A total of eight IBC patient tissues pre- and post-chemotherapy were quantified. p-values represent the difference between normal and pre-chemo and post-chemo, respectively. Full article ">Figure 5
Correlation of active RIPK2 expression with: (a) primary tumor stage; (b) presence of distant metastasis stage; (c) cancer stage; and (d) body mass index (BMI) in breast cancer. Active RIPK2 denotes autophosphorylation at site Y474. Full article ">Figure 6
Total RIPK2 mRNA expression in Non-IBC and IBC: (a) cell lines (n = 12) GEO (Gene Expression Omnibus dataset) (GSE40464) [<a href="#B62-cancers-10-00184" class="html-bibr">62</a>] and (b) tumor tissue (n = 40) GEO dataset (GSE45584) [<a href="#B63-cancers-10-00184" class="html-bibr">63</a>] from public breast cancer expression array datasets. IBC cell lines include MDA-IBC-3, MDA-IBC-2, SUM149, and SUM190, non-IBC includes MDA-MB-231, MDA-MB-468. In (b) Non-IBC mainly refers to Luminal A, Luminal B, HER2 overexpressed, and TNBC. Full article ">Figure 7
Correlation of active RIPK2 expression with HER2 mRNA expression: (a) and Ras association domain family protein 1A (RASSF1A) CpG methylation percentage; (b) correlation of RASSF1A mRNA expression and RASSF1A CpG methylation percentage; and (c) in IBC. CpG methylation analysis was carried out as described elsewhere [<a href="#B67-cancers-10-00184" class="html-bibr">67</a>] with focus on 32 CpG residues before the transcriptional start site (32 CpG x-axis label I (b) and CpG 13–32 (20 CpG) from the transcription start site. Full article ">Figure 8
Potential importance of RIPK2 in IBC. IBC cell line and patient samples reveal elevated levels of active RIPK2 that may be linked to epigenetic silencing of RASSF1A. Loss of RASSF1A, therefore, could be a new risk factor in IBC while active RIPK2 may have a role in regulating cellular response to chemotherapy. We speculate that RIPK2 inhibitors may be emerging therapeutic options for IBC. Full article ">

17 pages, 1227 KiB

Open AccessFeature PaperEditor’s ChoiceReview

Clinico-Pathological Importance of TGF-β/Phospho-Smad Signaling during Human Hepatic Fibrocarcinogenesis

by Katsunori Yoshida, Koichi Matsuzaki, Miki Murata, Takashi Yamaguchi, Kanehiko Suwa and Kazuichi Okazaki

Cancers 2018, 10(6), 183; https://doi.org/10.3390/cancers10060183 - 5 Jun 2018

Cited by 63 | Viewed by 8262

Abstract

Chronic viral hepatitis is a global public health problem, with approximately 570 million persons chronically infected. Hepatitis B and C viruses increase the risk of morbidity and mortality from liver cirrhosis, hepatocellular carcinoma (HCC), and extrahepatic complications that develop. Hepatitis virus infection induces [...] Read more.

Chronic viral hepatitis is a global public health problem, with approximately 570 million persons chronically infected. Hepatitis B and C viruses increase the risk of morbidity and mortality from liver cirrhosis, hepatocellular carcinoma (HCC), and extrahepatic complications that develop. Hepatitis virus infection induces transforming growth factor (TGF)-β, which influences microenvironments within the infected liver. TGF-β promotes liver fibrosis by up-regulating extracellular matrix production by hepatic stellate cells. TGF-β is also up-regulated in patients with HCC, in whom it contributes importantly to bringing about a favorable microenvironment for tumor growth. Thus, TGF-β is thought to be a major factor regulating liver fibrosis and carcinogenesis. Since TGF-β carries out regulatory signaling by influencing the phosphorylation of Smads, we have generated several kinds of phospho-specific antibodies to Smad2/3. Using these, we have identified three types of phospohorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C), linker phosphorylated Smad2/3 (pSmad2L and pSmad3L), and dually phosphorylated Smad3 (pSmad2L/C and pSmad3L/C). TGF-β-mediated pSmad2/3C signaling terminates cell proliferation; on the other hand, cytokine-induced pSmad3L signaling accelerates cell proliferation and promotes fibrogenesis. This review addresses TGF-β/Smad signal transduction in chronic liver injuries and carcinogenic processes. We also discuss the reversibility of Smad signaling after antiviral therapy. Full article

(This article belongs to the Special Issue TGF-Beta Signaling in Cancer)

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Figure 1
Differential phospho-Smad signals between tumor suppression and fibrocarcinogenesis. (A) Activated transforming growth factor (TGF)-β type I receptor (TβRI) phosphorylates COOH-tail serine residues of Smad2 and Smad3. Both COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) translocate with Smad4 to the nuclei of quiescent hepatocytes after regeneration. Smad2/3/4, complex binds the p21waf1 promoter and suppresses cell growth; (B) Pro-inflammatory cytokines (CK) such as tumor necrosis factor-α activate c-Jun N-terminal kinase (JNK), which phosphorylates the linker regions of Smad2 and Smad3. Linker phosphorylated Smad3 (PSmad3L) translocates with Smad4 to the nucleus and binds plasminogen activator inhibitor type 1 (PAI-1) promoter. Linker phosphorylated Smad2 (PSmad2L) is localized in the cytoplasm, and Smad2 translocates to the nucleus only after COOH-tail phosphorylation by TβRI. PSmad2L/C in cooperation with pSmad3L and Smad4 stimulate PAI-1 transcription and extracellular matrix (ECM) deposition. PSmad3L up-regulates c-Myc and stimulates cell growth, while suppressing the pSmad3C-mediated tumor suppressive pathway. Full article ">Figure 2
Phenotypic alternations of hepatocytes and HSC during the fibrocarcinogenic process in human chronic liver diseases. Quiescent hepatic stellate cells (HSC) are characterized by retinoid droplets in the cytoplasm and maintain liver homeostasis. HSC undergo constitutive activation to become myofibroblasts (MFB)-like cells after liver injury. MFB persistently produce an extracellular matrix (ECM) and induce liver fibrosis. The contraction of MFB contributes to increased portal resistance during liver fibrosis that presumably is reversible until the thickened septae, intrahepatic shunts, and lobular distortion that are characteristic of cirrhosis development, leading to fixed increases in portal pressure. Chronic liver damage promotes recurrent cycles of cellular proliferation, inflammation, fibrosis, and carcinogenesis. In pre-neoplastic hepatocytes, several growth factors and cytokines activate proliferation and invasion. As human hepatitis virus-related chronic liver diseases progress, chronic inflammation and hepatitis virus additively accelerate liver fibrosis and increase the risk of hepatocellular carcinoma (HCC). Genetic and epigenetic changes in the liver result in carcinogenesis. Effective antiviral therapy can reverse the pre-neoplastic properties of hepatocytes to a tumor-suppressive mode before the occurrence of genetic mutations that have been implicated for HCC occurrence. Full article ">

37 pages, 3829 KiB

Open AccessEditor’s ChoiceReview

Cancer Metastases to Bone: Concepts, Mechanisms, and Interactions with Bone Osteoblasts

by Alison B. Shupp, Alexus D. Kolb, Dimpi Mukhopadhyay and Karen M. Bussard

Cancers 2018, 10(6), 182; https://doi.org/10.3390/cancers10060182 - 4 Jun 2018

Cited by 95 | Viewed by 9813

Abstract

The skeleton is a unique structure capable of providing support for the body. Bone resorption and deposition are controlled in a tightly regulated balance between osteoblasts and osteoclasts with no net bone gain or loss. However, under conditions of disease, the balance between [...] Read more.

The skeleton is a unique structure capable of providing support for the body. Bone resorption and deposition are controlled in a tightly regulated balance between osteoblasts and osteoclasts with no net bone gain or loss. However, under conditions of disease, the balance between bone resorption and deposition is upset. Osteoblasts play an important role in bone homeostasis by depositing new bone osteoid into resorption pits. It is becoming increasingly evident that osteoblasts additionally play key roles in cancer cell dissemination to bone and subsequent metastasis. Our laboratory has evidence that when osteoblasts come into contact with disseminated breast cancer cells, the osteoblasts produce factors that initially reduce breast cancer cell proliferation, yet promote cancer cell survival in bone. Other laboratories have demonstrated that osteoblasts both directly and indirectly contribute to dormant cancer cell reactivation in bone. Moreover, we have demonstrated that osteoblasts undergo an inflammatory stress response in late stages of breast cancer, and produce inflammatory cytokines that are maintenance and survival factors for breast cancer cells and osteoclasts. Advances in understanding interactions between osteoblasts, osteoclasts, and bone metastatic cancer cells will aid in controlling and ultimately preventing cancer cell metastasis to bone. Full article

(This article belongs to the Special Issue Targeting Bone Metastasis in Cancer)

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Figure 1
Anatomy of long bones. Depicted are the three regions of long bones: epiphysis, metaphysis, and diaphysis. The outside of the bone is composed of dense cortical bone, while trabecular bone can be found in the interior and near bone ends. Also indicated are the growth plates, at the ends of the bone, and the sinusoidal vasculature that is found in the epiphysis. Full article ">Figure 2
Osteoblast differentiation. Murine osteoblast differentiation is characterized by three stages of growth marked by specific factor expression. During osteoblast proliferation (up to approximately 9 days), osteoblasts produce type I collagen. Osteoblasts enter early differentiation, or extracellular matrix maturation, at approximately 12 days of age, and express the proteins alkaline phosphatase, osteocalcin, and bone sialoprotein. Extracellular matrix mineralization occurs when osteoblasts are approximately 25 days old, where osteoblasts express the proteins osteonectin, osteocalcin, and osteopontin. Full article ">Figure 3
Stages of disease progression during bone metastatic cancer. Bone metastatic cancers may be broadly defined by three overarching stages: early disease, disease progression, and advanced or late stage disease. During early stage disease, disseminated tumor cells circulating in the vasculature enter bone and seed mainly as single cells. Cells may be undetectable by current technological methods due to their solitary nature. Cancer cell dormancy may also occur. As bone metastatic disease progresses over a period of months to years to potentially decades, dormant cancer cells become re-awakened leading to proliferation and coalescing of smaller, micrometastatic lesions. Lesions may become detectable by current technological methods. Treatments to reduce tumor size and alter bone remodeling may occur. Over time, tumors may become refractory to treatment modalities, leading to sustained tumor cell proliferation and excessive tumor burden. Macrometastatic lesions form. Patients may experience effects of increased tumor burden including severe bone pain, fractures, and hypercalcemia. Patient quality of life progressively deteriorates. Treatment modalities during advanced stage disease are mainly palliative to reduce complications from excessive bone tumor burden. Full article ">Figure 4
Tumor-educated osteoblast cells have altered cytokine production when compared to untreated osteoblasts. Conditioned media was prepared from untreated MC3T3-E1 osteoblasts or TEO cells and subjected to a RayBiotech Quantibody® Quantitative Multiplex ELISA Array. Three separate batches of conditioned media were assayed per condition. Shown are the mean protein concentration of Fractalkine and Axl produced by either untreated MC3T3-E1 osteoblasts or TEO cells. * p < 0.05. Full article ">Figure 5
Mice inoculated with a mix of TEO cells plus MDA-MB-231 breast cancer cells lived longer and had smaller tumors than mice inoculated with a mix of untreated MC3T3-E1 cells plus MDA-MB-231 breast cancer cells, or breast cancer cells inoculated alone. Athymic nude mice were inoculated with either mixes of (1) TEO cells plus MDA-MB-231GFP breast cancer cells; (2) MC3T3-E1 osteoblasts plus MDA-MB-231GFP breast cancer cells; or (3) MDA-MB-231GFP breast cancer cells alone. (A) Kaplain-Meier Survival curve of percent mouse survival over time. Blue line, TEO plus MDA-MB-231GFP; red line, MC3T3-E1 plus MDA-MB-231GFP; green line, MDA-MB-231 cells alone. (B) Tumor size (mm3). At least four mice were used per condition per time point. Shown are representative examples. Full article ">Figure 6
The ‘Vicious Cycle’ of bone degradation. In the ‘vicious cycle’ of cancer metastasis to bone, metastatic cancer cells produce parathyroid hormone related protein (PTHrP), interleukin-8 (IL-8), interleukin-11 (IL-11), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2) (lytic and blastic cancers) as well as bone morphogenic proteins (BMPs), endothelin-1 (ET-1), dickkopf-1 (DKK-1), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) (blastic cancers). These factors promote increased osteoblast production of interleukin-6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), growth related oncogene-alpha (GRO-alpha), IL-11, and COX-2 that act as chemoattractants, maintenance, and survival factors for lytic metastatic cancer cells. Growth factors, including VEGF, insulin growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and tumor-growth factor-beta (TGF-beta) as produced by osteoblasts serve to promote proliferation of blastic cancers. Osteoblasts also produce increased amounts of soluble receptor activator of nuclear factor kappa beta-ligand (RANK-L), which binds to RANK receptors on pre-osteoclasts to initiate osteoclastogenesis. Furthermore, breast cancer cells that express Jagged1 ligand bind to the Notch receptor on osteoblasts leading to increased osteoblast-derived RANK-L expression and increased osteoclast activation. Jagged1 expressing breast cancer cells can also bind to the Notch receptor on pre-osteoclasts and initiate osteoclastogenesis independent of RANK-L. Activated osteoclasts resorb bone by secretion of proteases including cathepsin K into resorption pits, and release TGF-beta and IGF-1 from the bone matrix. TGF-beta and IGF-1 act on metastatic tumor cells to promote proliferation and continued expression of PTHrP, IL-8, IL-11, PGE2, COX-2, BMPs, ET-1, DKK-1, PDGF, and EGF from cancer cells. Full article ">

16 pages, 916 KiB

Open AccessReview

The Glucose-Regulated MiR-483-3p Influences Key Signaling Pathways in Cancer

by Felice Pepe, Rosa Visone and Angelo Veronese

Cancers 2018, 10(6), 181; https://doi.org/10.3390/cancers10060181 - 4 Jun 2018

Cited by 34 | Viewed by 6110

Abstract

The hsa-mir-483 gene, located within the IGF2 locus, transcribes for two mature microRNAs, miR-483-5p and miR-483-3p. This gene, whose regulation is mediated by the the CTNNB1/USF1 complex, shows an independent expression from its host gene IGF2. The miR-483-3p affects the Wnt/β-catenin, [...] Read more.

The hsa-mir-483 gene, located within the IGF2 locus, transcribes for two mature microRNAs, miR-483-5p and miR-483-3p. This gene, whose regulation is mediated by the the CTNNB1/USF1 complex, shows an independent expression from its host gene IGF2. The miR-483-3p affects the Wnt/β-catenin, the TGF-β, and the TP53 signaling pathways by targeting several genes as CTNNB1, SMAD4, IGF1, and BBC3. Accordingly, miR-483-3p is associated with various tissues specific physiological properties as insulin and melanin production, as well as with cellular physiological functions such as wounding, differentiation, proliferation, and survival. Deregulation of miR-483-3p is observed in different types of cancer, and its overexpression can inhibit the pro-apoptotic pathway induced by the TP53 target effectors. As a result, the oncogenic characteristics of miR-483-3p are linked to the effect of some of the most relevant cancer-related genes, TP53 and CTNNB1, as well as to one of the most important cancer hallmark: the aberrant glucose metabolism of tumor cells. In this review, we summarize the recent findings regarding the miR-483-3p, to elucidate its functional role in physiological and pathological contexts, focusing overall on its involvement in cancer and in the TP53 pathway. Full article

(This article belongs to the Special Issue p53 Signaling in Cancers)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Stem-loop sequence of the hsa-miR-483 and mature miRNAs. In the Figure are reported genomic position, sequence of the hsa-miR-483 gene. Sequences data from miRBase database [<a href="#B27-cancers-10-00181" class="html-bibr">27</a>,<a href="#B28-cancers-10-00181" class="html-bibr">28</a>,<a href="#B29-cancers-10-00181" class="html-bibr">29</a>,<a href="#B30-cancers-10-00181" class="html-bibr">30</a>,<a href="#B31-cancers-10-00181" class="html-bibr">31</a>]. Full article ">Figure 2
Representation of miR-483-3p feedback with the USF1/CTNNB1 complex. (a) Physiological negative feedback; (b) CTNNB1 mutated is no more regulated by miR-483-3p, which expression increase. Full article ">Figure 3
Schematic representation of the role of O-linked β-N-acetylglucosamine transferase (OGT) and miR-483-3p in the miR-145-5p/TP53 axis in hepatocellular carcinoma (HCC). In the Figure are showed proteins (squares) and miRNAs (ovals) and their functional connection involved in the miR-145-5p/TP53 axis in HCC. Briefly, miR-145-5p targets MDM2 and reduces expression of miR-483-3p, permitting the activation of TP53 that induce PUMA and apoptosis. In case of OGT over-activity, miR-145-5p is unable to regulate miR-483-3p which expression is increased by CTNNB1 activity. This leads the miR-483-3p to target PUMA and blocking the pro-apoptotic effect of TP53. Full article ">

28 pages, 5341 KiB

Open AccessArticle

KIAA0100 Modulates Cancer Cell Aggression Behavior of MDA-MB-231 through Microtubule and Heat Shock Proteins

by Zhenyu Zhong, Vaishali Pannu, Matthew Rosenow, Adam Stark and David Spetzler

Cancers 2018, 10(6), 180; https://doi.org/10.3390/cancers10060180 - 4 Jun 2018

Cited by 10 | Viewed by 4375

Abstract

The KIAA0100 gene was identified in the human immature myeloid cell line cDNA library. Recent studies have shown that its expression is elevated in breast cancer and associated with more aggressive cancer types as well as poor outcomes. However, its cellular and molecular [...] Read more.

The KIAA0100 gene was identified in the human immature myeloid cell line cDNA library. Recent studies have shown that its expression is elevated in breast cancer and associated with more aggressive cancer types as well as poor outcomes. However, its cellular and molecular function is yet to be understood. Here we show that silencing KIAA0100 by siRNA in the breast cancer cell line MDA-MB-231 significantly reduced the cancer cells’ aggressive behavior, including cell aggregation, reattachment, cell metastasis and invasion. Most importantly, silencing the expression of KIAA0100 particularly sensitized the quiescent cancer cells in suspension culture to anoikis. Immunoprecipitation, mass spectrometry and immunofluorescence analysis revealed that KIAA0100 may play multiple roles in the cancer cells, including stabilizing microtubule structure as a microtubule binding protein, and contributing to MDA-MB-231 cells Anoikis resistance by the interaction with stress protein HSPA1A. Our study also implies that the interaction between KIAA0100 and HSPA1A may be targeted for new drug development to specifically induce anoikis cell death in the cancer cell. Full article

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