Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several ca... more Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several cancer types, and MET has been suggested to interact with stem cell networks. In vitro studies have further suggested a possible benefit of a combined treatment using PARP and MET inhibitors. We used a tissue microarray (TMA) with 130 samples of advanced-stage high-grade serous fallopian tube/ovarian cancer (HGSC) to investigate the prognostic value of MET protein expression alone and in combination with the stem cell factor SOX2. The possible synergistic effects of a PARP and MET inhibitor treatment were evaluated in two cell lines with BRCA1 or BRCA2 deficiency and in their BRCA1/2-proficient counterparts. Patients with tumors positive for MET had worse overall survival (log-rank test, p = 0.015) compared to patients with MET-negative tumors. The prognostic role of MET was even more prominent in the subgroup of patients with SOX2-negative tumors (p = 0.0081). No synergistic effects of th...
Additional file 6. Figure showing estradiol and tamoxifen response in the ZR-75-1, T47D and EFM-1... more Additional file 6. Figure showing estradiol and tamoxifen response in the ZR-75-1, T47D and EFM-19 models. Relative proliferation of parental and fulvestrant-resistant cells in estrogen depleted (A) or normal (B) growth media with or without supplementation with 1 nM estradiol (E2) (A) or 100 nM 4-hydroxytamoxifen (4-OHT) (B) for 6 days. Each graph represents combined data (average ± SEM) from two biological experiments with three technical replicates each. Statistical differences were determined using one-way ANOVA with Tukey's post-hoc test. *** represents p-value ≤0.0001, ** ≤0.001, ns represents no statistical differences. Stars and 'ns' in (A) indicate statistical differences compared to -E2 for each cell model unless indicated otherwise. Stars and 'ns' in (B) indicate statistical differences compared to -4-OHT for each cell model unless indicated otherwise.
Additional file 2. Table showing patient characteristics for patient samples pre and post fulvest... more Additional file 2. Table showing patient characteristics for patient samples pre and post fulvestrant treatment.
Additional file 3. Figure showing that fulvestrant-resistant cells proliferate in the presence of... more Additional file 3. Figure showing that fulvestrant-resistant cells proliferate in the presence of fulvestrant and downregulate ER signaling. A) Time in weeks for each parental cell line to develop resistance to fulvestrant, from initial 100 pM dose until able to proliferate in presence of 100 nM fulvestrant. B) Proliferation curves for parental (-P, black lines) and fulvestrant-resistant (-FR, red lines) cells in the absence (ctrl, solid lines) or presence (+F, dotted lines) of 100 nM fulvestrant assessed using SRB assays (CAMA-1, MCF7, HCC1428, ZR-75-1) or xCELLigence system (T47D, EFM-19). Graphs represent combined data (average ± SEM) from two (xCELLigence) or three (SRB) biological experiments with at least three technical replicates each. Statistical differences were determined with two-way ANOVA and Tukey's post-hoc test. * represents p-value ≤0.01, ** ≤0.001 and *** ≤0.0001 between fulvestrant-treated parental (black dotted lines) and fulvestrant-resistant (red dotted lin...
Additional file 7. Figure showing BAF and LogR files for copy number data. B allele frequency (BA... more Additional file 7. Figure showing BAF and LogR files for copy number data. B allele frequency (BAF) and LogR genome-wide plots for SNP profiled cell line data presented in Fig. 4a. Chromosomes are ordered along the x-axis from 1 (left) to Y (right). Green lines represent segments derived from ASCAT 2 segmentation of the data.
Additional file 1. Table showing normalized gene expression data from parental and fulvestrant-re... more Additional file 1. Table showing normalized gene expression data from parental and fulvestrant-resistant CAMA-1, MCF7, T47D and HCC1428 cell lines.
CD44 is stained with Permanent Red and CD24 with diaminobenzidene (DAB). Magnification × 20. A tu... more CD44 is stained with Permanent Red and CD24 with diaminobenzidene (DAB). Magnification × 20. A tumor positive for both CD44/CD24(white arrow) and CD44/CD24(black arrow) cancer cells, although the predominant phenotype is CD44/CD24. Almost all cells in this tumor are CD44/CD24. No CD24 staining is seen. A tumor with predominantly CD44/CD24cells. A few CD44/CD24cells are also present (black arrow). A tumor positive for the CD44/CD24phenotype. No CD44 staining is present.<b>Copyright information:</b>Taken from "The CD44/CD24phenotype is enriched in basal-like breast tumors"http://breast-cancer-research.com/content/10/3/R53Breast Cancer Research : BCR 2008;10(3):R53-R53.Published online 17 Jun 2008PMCID:PMC2481503.
CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in est... more CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination ...
Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceoso... more Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.
Endocrine therapy is the mainstay of treatment for metastatic estrogen receptor (ER) positive bre... more Endocrine therapy is the mainstay of treatment for metastatic estrogen receptor (ER) positive breast cancer. The selective ER degrader (SERD) fulvestrant is increasingly relevant due to its high therapeutic efficacy and often used in combination with other targeted treatments in the metastatic setting. However, most metastatic patients inevitably progress during therapy and develop resistance, constituting a major clinical problem. Our aim is to identify mechanisms conferring resistance to fulvestrant and evaluate potential therapeutic interventions. We have generated several fulvestrant-resistant (FR) in vitro models through chronic exposure of established breast cancer cell lines (i.e. MCF7, Cama1, T47D and HCC1428) to increasing concentrations of fulvestrant. The models have been characterized by assessing gene expression alterations using microarrays, protein expression by western blots, cell cycle profiling by flow cytometry and drug sensitivity by proliferation assays. These e...
Background Estrogen receptor (ER) is a main driver of tumor progression in ER+ metastatic breast ... more Background Estrogen receptor (ER) is a main driver of tumor progression in ER+ metastatic breast cancer (MBC). The use of endocrine therapy can effectively control the disease in a large proportion of patients. However, the majority of MBC eventually become resistant and progress. To elucidate the mechanisms of acquired resistance to endocrine treatment is key in order to better select therapeutic partners and delay disease progression. Methods A panel of ER+ breast cancer cell lines initially sensitive to the selective estrogen receptor degrader (SERD) fulvestrant was exposed to increasing concentrations of this drug over several months to induce resistance. Cell proliferation was determined with the xCELLigence system. Protein expression was measured by phospho-kinase array and western blotting. RNA expression was evaluated by gene expression microarray analysis (Illumina) and validated by RT-qPCR. Cell cycle distribution was analyzed by flow cytometry. Results Using an unbiased a...
Clustering was based on 364 genes from the intrinsic gene list published by Sørlie and colleagues... more Clustering was based on 364 genes from the intrinsic gene list published by Sørlie and colleagues [] that matched our cDNA clones. Colored boxes indicate classification of each tumor into subtypes/subgroups. Filled or open boxes indicate the percentage of cells in each tumor positive for the CD44/CD24and CD44/CD24phenotypes as determined by immunohistochemistry. SR, steroid receptor. Hu classification, Hu and colleagues [].<b>Copyright information:</b>Taken from "The CD44/CD24phenotype is enriched in basal-like breast tumors"http://breast-cancer-research.com/content/10/3/R53Breast Cancer Research : BCR 2008;10(3):R53-R53.Published online 17 Jun 2008PMCID:PMC2481503.
Faculty of Medicine Doctoral Dissertation Series, 2008
Stem cells have qualities that clearly distinguish them from all other cells. Capabilities like s... more Stem cells have qualities that clearly distinguish them from all other cells. Capabilities like self-renewal, differentiation and migration make them truly powerful. This thesis deals with different aspects of stem/progenitor cells in relation to cancer. Certain types of neural progenitor cells (NPCs) have been shown to possess a potential of tracking glioma cells in the brain. This makes them potentially very interesting as delivery vehicles in glioma therapy. In the first part of this thesis, we showed that certain NPCs have an ability of inhibiting tumor growth. We inoculated progenitor cells together with glioma cells in the nucleus caudatus of Fischer rats and saw a prolonged survival of the animals. We further demonstrated that we could enhance migration of these tumor-inhibitory NPCs to the site of glioma growth in a chemokine-dependent fashion. We introduced the chemokine receptor CXCR3 in the NPCs, and showed an enhanced migration of such overexpressing NPCs over the corpus callosum towards the glioma when inoculated at a distance from the tumor. In the second part of the thesis, we studied tumor-initiating cells in breast cancer. Tumors consist of a variety of cells with different features. A small population of cancer stem cells is believed to maintain this diversity. In breast cancer, a subpopulation of CD44+/CD24- cells is enriched for tumorigenic ability. We have stained human breast tumors for these markers and demonstrated a correlation of CD44+/CD24- tumor cells to basal-like and BRCA1 hereditary breast cancer. We also saw an increase of cells with the CD44+/CD24- phenotype when growing breast cancer cell lines with basal-like characteristics in non-adherent spherical clusters (mammospheres). Growing cells under such conditions enrich for cells with stem cell properties, as indicated by decreased proliferative rate and enhanced ability to generate new spheres from one single cell. We further showed an enhanced resistance to chemotherapeutic drugs for mammosphere-derived cells. In conclusion, stem and tumor cells seem to be linked in many ways, and increasing knowledge of their interactions could hopefully in the future lead to improved therapies against cancer. (Less)
Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several ca... more Overexpression of the receptor tyrosine kinase MET has been linked to poor survival in several cancer types, and MET has been suggested to interact with stem cell networks. In vitro studies have further suggested a possible benefit of a combined treatment using PARP and MET inhibitors. We used a tissue microarray (TMA) with 130 samples of advanced-stage high-grade serous fallopian tube/ovarian cancer (HGSC) to investigate the prognostic value of MET protein expression alone and in combination with the stem cell factor SOX2. The possible synergistic effects of a PARP and MET inhibitor treatment were evaluated in two cell lines with BRCA1 or BRCA2 deficiency and in their BRCA1/2-proficient counterparts. Patients with tumors positive for MET had worse overall survival (log-rank test, p = 0.015) compared to patients with MET-negative tumors. The prognostic role of MET was even more prominent in the subgroup of patients with SOX2-negative tumors (p = 0.0081). No synergistic effects of th...
Additional file 6. Figure showing estradiol and tamoxifen response in the ZR-75-1, T47D and EFM-1... more Additional file 6. Figure showing estradiol and tamoxifen response in the ZR-75-1, T47D and EFM-19 models. Relative proliferation of parental and fulvestrant-resistant cells in estrogen depleted (A) or normal (B) growth media with or without supplementation with 1 nM estradiol (E2) (A) or 100 nM 4-hydroxytamoxifen (4-OHT) (B) for 6 days. Each graph represents combined data (average ± SEM) from two biological experiments with three technical replicates each. Statistical differences were determined using one-way ANOVA with Tukey's post-hoc test. *** represents p-value ≤0.0001, ** ≤0.001, ns represents no statistical differences. Stars and 'ns' in (A) indicate statistical differences compared to -E2 for each cell model unless indicated otherwise. Stars and 'ns' in (B) indicate statistical differences compared to -4-OHT for each cell model unless indicated otherwise.
Additional file 2. Table showing patient characteristics for patient samples pre and post fulvest... more Additional file 2. Table showing patient characteristics for patient samples pre and post fulvestrant treatment.
Additional file 3. Figure showing that fulvestrant-resistant cells proliferate in the presence of... more Additional file 3. Figure showing that fulvestrant-resistant cells proliferate in the presence of fulvestrant and downregulate ER signaling. A) Time in weeks for each parental cell line to develop resistance to fulvestrant, from initial 100 pM dose until able to proliferate in presence of 100 nM fulvestrant. B) Proliferation curves for parental (-P, black lines) and fulvestrant-resistant (-FR, red lines) cells in the absence (ctrl, solid lines) or presence (+F, dotted lines) of 100 nM fulvestrant assessed using SRB assays (CAMA-1, MCF7, HCC1428, ZR-75-1) or xCELLigence system (T47D, EFM-19). Graphs represent combined data (average ± SEM) from two (xCELLigence) or three (SRB) biological experiments with at least three technical replicates each. Statistical differences were determined with two-way ANOVA and Tukey's post-hoc test. * represents p-value ≤0.01, ** ≤0.001 and *** ≤0.0001 between fulvestrant-treated parental (black dotted lines) and fulvestrant-resistant (red dotted lin...
Additional file 7. Figure showing BAF and LogR files for copy number data. B allele frequency (BA... more Additional file 7. Figure showing BAF and LogR files for copy number data. B allele frequency (BAF) and LogR genome-wide plots for SNP profiled cell line data presented in Fig. 4a. Chromosomes are ordered along the x-axis from 1 (left) to Y (right). Green lines represent segments derived from ASCAT 2 segmentation of the data.
Additional file 1. Table showing normalized gene expression data from parental and fulvestrant-re... more Additional file 1. Table showing normalized gene expression data from parental and fulvestrant-resistant CAMA-1, MCF7, T47D and HCC1428 cell lines.
CD44 is stained with Permanent Red and CD24 with diaminobenzidene (DAB). Magnification × 20. A tu... more CD44 is stained with Permanent Red and CD24 with diaminobenzidene (DAB). Magnification × 20. A tumor positive for both CD44/CD24(white arrow) and CD44/CD24(black arrow) cancer cells, although the predominant phenotype is CD44/CD24. Almost all cells in this tumor are CD44/CD24. No CD24 staining is seen. A tumor with predominantly CD44/CD24cells. A few CD44/CD24cells are also present (black arrow). A tumor positive for the CD44/CD24phenotype. No CD44 staining is present.<b>Copyright information:</b>Taken from "The CD44/CD24phenotype is enriched in basal-like breast tumors"http://breast-cancer-research.com/content/10/3/R53Breast Cancer Research : BCR 2008;10(3):R53-R53.Published online 17 Jun 2008PMCID:PMC2481503.
CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in est... more CDK4/6 inhibitors (CDK4/6i) combined with endocrine therapy have shown impressive efficacy in estrogen receptor-positive advanced breast cancer. However, most patients will eventually experience disease progression on this combination, underscoring the need for effective subsequent treatments or better initial therapies. Here, we show that triple inhibition with fulvestrant, CDK4/6i and AKT inhibitor (AKTi) durably impairs growth of breast cancer cells, prevents progression and reduces metastasis of tumor xenografts resistant to CDK4/6i-fulvestrant combination or fulvestrant alone. Importantly, switching from combined fulvestrant and CDK4/6i upon resistance to dual combination with AKTi and fulvestrant does not prevent tumor progression. Furthermore, triple combination with AKTi significantly inhibits growth of patient-derived xenografts resistant to combined CDK4/6i and fulvestrant. Finally, high phospho-AKT levels in metastasis of breast cancer patients treated with a combination ...
Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceoso... more Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.
Endocrine therapy is the mainstay of treatment for metastatic estrogen receptor (ER) positive bre... more Endocrine therapy is the mainstay of treatment for metastatic estrogen receptor (ER) positive breast cancer. The selective ER degrader (SERD) fulvestrant is increasingly relevant due to its high therapeutic efficacy and often used in combination with other targeted treatments in the metastatic setting. However, most metastatic patients inevitably progress during therapy and develop resistance, constituting a major clinical problem. Our aim is to identify mechanisms conferring resistance to fulvestrant and evaluate potential therapeutic interventions. We have generated several fulvestrant-resistant (FR) in vitro models through chronic exposure of established breast cancer cell lines (i.e. MCF7, Cama1, T47D and HCC1428) to increasing concentrations of fulvestrant. The models have been characterized by assessing gene expression alterations using microarrays, protein expression by western blots, cell cycle profiling by flow cytometry and drug sensitivity by proliferation assays. These e...
Background Estrogen receptor (ER) is a main driver of tumor progression in ER+ metastatic breast ... more Background Estrogen receptor (ER) is a main driver of tumor progression in ER+ metastatic breast cancer (MBC). The use of endocrine therapy can effectively control the disease in a large proportion of patients. However, the majority of MBC eventually become resistant and progress. To elucidate the mechanisms of acquired resistance to endocrine treatment is key in order to better select therapeutic partners and delay disease progression. Methods A panel of ER+ breast cancer cell lines initially sensitive to the selective estrogen receptor degrader (SERD) fulvestrant was exposed to increasing concentrations of this drug over several months to induce resistance. Cell proliferation was determined with the xCELLigence system. Protein expression was measured by phospho-kinase array and western blotting. RNA expression was evaluated by gene expression microarray analysis (Illumina) and validated by RT-qPCR. Cell cycle distribution was analyzed by flow cytometry. Results Using an unbiased a...
Clustering was based on 364 genes from the intrinsic gene list published by Sørlie and colleagues... more Clustering was based on 364 genes from the intrinsic gene list published by Sørlie and colleagues [] that matched our cDNA clones. Colored boxes indicate classification of each tumor into subtypes/subgroups. Filled or open boxes indicate the percentage of cells in each tumor positive for the CD44/CD24and CD44/CD24phenotypes as determined by immunohistochemistry. SR, steroid receptor. Hu classification, Hu and colleagues [].<b>Copyright information:</b>Taken from "The CD44/CD24phenotype is enriched in basal-like breast tumors"http://breast-cancer-research.com/content/10/3/R53Breast Cancer Research : BCR 2008;10(3):R53-R53.Published online 17 Jun 2008PMCID:PMC2481503.
Faculty of Medicine Doctoral Dissertation Series, 2008
Stem cells have qualities that clearly distinguish them from all other cells. Capabilities like s... more Stem cells have qualities that clearly distinguish them from all other cells. Capabilities like self-renewal, differentiation and migration make them truly powerful. This thesis deals with different aspects of stem/progenitor cells in relation to cancer. Certain types of neural progenitor cells (NPCs) have been shown to possess a potential of tracking glioma cells in the brain. This makes them potentially very interesting as delivery vehicles in glioma therapy. In the first part of this thesis, we showed that certain NPCs have an ability of inhibiting tumor growth. We inoculated progenitor cells together with glioma cells in the nucleus caudatus of Fischer rats and saw a prolonged survival of the animals. We further demonstrated that we could enhance migration of these tumor-inhibitory NPCs to the site of glioma growth in a chemokine-dependent fashion. We introduced the chemokine receptor CXCR3 in the NPCs, and showed an enhanced migration of such overexpressing NPCs over the corpus callosum towards the glioma when inoculated at a distance from the tumor. In the second part of the thesis, we studied tumor-initiating cells in breast cancer. Tumors consist of a variety of cells with different features. A small population of cancer stem cells is believed to maintain this diversity. In breast cancer, a subpopulation of CD44+/CD24- cells is enriched for tumorigenic ability. We have stained human breast tumors for these markers and demonstrated a correlation of CD44+/CD24- tumor cells to basal-like and BRCA1 hereditary breast cancer. We also saw an increase of cells with the CD44+/CD24- phenotype when growing breast cancer cell lines with basal-like characteristics in non-adherent spherical clusters (mammospheres). Growing cells under such conditions enrich for cells with stem cell properties, as indicated by decreased proliferative rate and enhanced ability to generate new spheres from one single cell. We further showed an enhanced resistance to chemotherapeutic drugs for mammosphere-derived cells. In conclusion, stem and tumor cells seem to be linked in many ways, and increasing knowledge of their interactions could hopefully in the future lead to improved therapies against cancer. (Less)
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