Loxl3 Promotes Melanoma Progression and Dissemination Influencing Cell Plasticity and Survival
"> Figure 1
<p>Strategy to generate mice with conditional melanocyte-specific targeting of <span class="html-italic">Loxl3</span>. (<b>A</b>) Schematic representation of the Loxl3 alleles present in the mice studied: wild-type <span class="html-italic">Loxl3<sup>wt</sup></span> allele, knockout first allele <span class="html-italic">Loxl3<sup>LacZ</sup></span> modified through Flippase to obtain a conditional <span class="html-italic">Loxl3<sup>loxP</sup></span> allele. See Materials and Methods for details. (<b>B</b>) Confirmation by diagnostic PCR of the genetic changes produced by CreERT2 recombinase in <span class="html-italic">Braf</span>, <span class="html-italic">Pten,</span> and <span class="html-italic">Loxl3</span> genes in genomic DNA from tails from <span class="html-italic">Braf<sup>+/+</sup> Pten<sup>+/+</sup> Loxl3<sup>+/+</sup></span> WT untreated mice (left panels), and tails and tumors from conditional <span class="html-italic">Braf<sup>V600E</sup> Pten<sup>loxP</sup> Loxl3<sup>loxP</sup></span> mice upon 4-HT treatment (right panels). The expected size in base pairs (bp) of the different PCR products from the corresponding alleles is indicated before each of them on the right side. (<b>C</b>) Schematic representation of the <span class="html-italic">Braf<sup>V600E</sup></span>-, <span class="html-italic">Pten<sup>loxP</sup></span>-, <span class="html-italic">Tyr:CreER<sup>T2</sup></span>-<span class="html-italic">,</span> and <span class="html-italic">Loxl3<sup>loxP</sup></span>-modified alleles included in the conditional mouse model. Allele nomenclature: <span class="html-italic">Braf<sup>V600E</sup></span>: <span class="html-italic">Braf</span> allele targeted with <span class="html-italic">Braf<sup>V600E</sup></span> modification; <span class="html-italic">Braf<sup>CA</sup></span>: constitutively active <span class="html-italic">Braf</span> allele upon CreERT2-mediated recombination; <span class="html-italic">Pten<sup>loxP</sup></span>: <span class="html-italic">Pten</span> allele modified to include loxP-flanked exon 5; <span class="html-italic">Pten<sup>KO</sup></span>: loss of function <span class="html-italic">Pten</span> allele upon CreERT2-mediated recombination; <span class="html-italic">Tyr:CreER<sup>T2</sup></span>: 4-HT conditionally active CreERT2 expressed under the control of the <span class="html-italic">Tyrosinase</span> (Tyr) promoter; <span class="html-italic">Loxl3<sup>loxP</sup></span>: <span class="html-italic">Loxl3</span> allele including loxP-flanked exon 3 after Flippase-induced recombination of <span class="html-italic">Loxl3<sup>lacZ</sup></span> allele; <span class="html-italic">Loxl3<sup>KO</sup></span>: loss of function <span class="html-italic">Loxl3</span> allele upon CreERT2-mediated recombination; wt denotes wild-type alleles. FRT: Flippase recognition sites; loxP: Cre recombinase recognition sites.</p> "> Figure 2
<p><span class="html-italic">Loxl3</span> targeting in a melanoma mouse model delays tumor development and progression. (<b>A</b>) Schematic representation of the protocol followed to induce melanoma development on the backs of <span class="html-italic">Braf Pten L3<sup>wt</sup></span> and <span class="html-italic">Braf Pten L3<sup>loxP</sup></span> mice. Representative pictures from the appearance and evolution of the lesions at indicated days upon tamoxifen treatment in <span class="html-italic">Braf Pten L3<sup>wt</sup></span> (Loxl3 WT) and <span class="html-italic">Braf Pten L3<sup>loxP</sup></span> (Loxl3 KO) mice are shown (lower panels). (<b>B</b>) Hematoxylin and eosin (H/E) staining of skin sections from Loxl3 WT and Loxl3 KO lesions. Blue frames denote the amplified areas shown in the middle and right side images. Scale bars: 500 µm (left pictures), 100 µm (middle and right pictures). (<b>C</b>) Tyrp2 staining by IHC of skin sections from Loxl3 WT and Loxl3 KO lesions. Scale bar: 100 µm. (<b>D</b>) Percentage (%) of animals free of lesions from <span class="html-italic">Braf Pten L3<sup>wt</sup></span> (Loxl3 WT) and <span class="html-italic">Braf Pten L3<sup>loxP</sup></span> (Loxl3 KO) backgrounds at different days upon 4-HT treatment. (<b>E</b>) Area of pigmented lesions in <span class="html-italic">Braf Pten L3<sup>wt</sup></span> (Loxl3 WT) and <span class="html-italic">Braf Pten L3<sup>loxP</sup></span> (Loxl3 KO) mice at different weeks upon 4-HT administration. (<b>F</b>) Kaplan–Meier survival curve of <span class="html-italic">Braf Pten L3<sup>wt</sup></span> (Loxl3 WT) and <span class="html-italic">Braf Pten L3<sup>loxP</sup></span> (Loxl3 KO) mice following 4-HT treatment. The <span class="html-italic">p</span>-values were calculated by Mantel–Cox test (<b>D</b>,<b>F</b>) and two-sided unpaired Student’s <span class="html-italic">t</span>-test (<b>E</b>); <span class="html-italic">n</span> indicates the number of monitored animals with the indicated genotype (<b>D</b>–<b>F</b>). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 3
<p>Loxl3 inactivation in melanocytes decreases melanoma lymphatic dissemination. (<b>A</b>) Representative images of proximal and distal lymph nodes collected at 6 weeks after 4-HT treatment from Loxl3 WT and Loxl3 KO mice in the <span class="html-italic">Braf Pten</span> genetic background. The image on the upper right shows lymph nodes collected from untreated age-matched control <span class="html-italic">Braf Pten L3<sup>wt</sup></span> animals. (<b>B</b>) H/E, Tyrp2, and Sox10 stainings of lymph node sections from <span class="html-italic">Braf Pten L3<sup>wt</sup></span> mice bearing 4-HT-induced melanomas. Blue frames indicate an amplified area from the middle sections depicted in the lower left quarters (middle and lower panels). White arrows point to cytoplasmic (Tyrp2) and nuclear (Sox10) detection. Scale bars: 100 µm. (<b>C</b>) Representative images (top) of Tyrp2 staining in proximal and distal lymph node sections from Loxl3 WT and proximal lymph node sections from Loxl3 KO animals bearing melanomas developed upon 4-HT administration. Black frames denote amplified sections shown on the left (Loxl3 WT) and right (Loxl3 WT) sides; some of them are rotated 90 °C regarding the original image. Scale bars: 200 µm. The chart (bottom) indicates the number and percentage of Tyrp2-positive distal and proximal lymph nodes detected in Loxl3 WT and KO mice sacrificed at 42 days after 4-HT treatment. (<b>D</b>) Quantification of total Tyrp2 stained area in lymph node sections collected from mice as detailed in (<b>A</b>). Lymph nodes from Loxl3 WT and KO mice (<span class="html-italic">n</span> = 7) were analyzed. The <span class="html-italic">p</span>-value was calculated by two-sided unpaired Student’s <span class="html-italic">t</span>-test. ** <span class="html-italic">p</span> < 0.01.</p> "> Figure 4
<p>Loxl3 silencing in primary melanoma-derived cells decreases proliferation and increases DNA damage. (<b>A</b>) Scheme showing the derivation and establishment of primary cell lines from Loxl3 WT and KO melanomas. (<b>B</b>) Characterization by protein expression analyses of melanoma-derived MeL3 cell line compared to human (A375P) and mouse (YUMM1.7) established melanoma cell lines. The 137d line represents a negative control of primary tumor-derived cells without the expected protein pattern regarding TYRP2, SOX10, pERK1/2, total ERK, and PTEN protein expression. Tubulin was used as a loading control. Note that the LOXL3Δ isoform described in human cell lines [<a href="#B38-cancers-14-01200" class="html-bibr">38</a>] is not detected in mouse cell lines. Uncropped Western Blots and densitometry analyses can be found at <a href="#app1-cancers-14-01200" class="html-app">supplementary original figures</a>. (<b>C</b>) Phase-contrast images of MeL3 cells upon Loxl3 silencing using two specific shRNAs (sh2 and sh3) and a nontargeting control (NTC) sequence. Scale bars: 100 µm. (<b>D</b>) Immunoblot analyses of melanoma markers (TYRP2, SOX10) and PTEN and BRAF status in control (NTC) and Loxl3-silenced (sh2 and sh3) MeL3 cells. Human.A375P melanoma cells were included as an additional control. GAPDH was used as a loading control. The pERK 1/2 relative to total ERK levels denote BRAF activation status (<b>B</b>,<b>D</b>); s.e.: short exposure, l.e.: long exposure. Uncropped Western Blots and densitometry analyses can be found at <a href="#app1-cancers-14-01200" class="html-app">supplementary original figures</a>. (<b>E</b>) Cell proliferation analyses of control (NTC) and Loxl3-silenced (sh2 and sh3) MeL3 cells at different time points after seeding represented as % of cell growth. (<b>F</b>) Annexin/PI staining of MeL3 control and Loxl3-silenced cells at the indicated time points after seeding represented as % of total cell population. No comparisons between cells, regardless of apoptosis stage, were significant. (<b>G</b>) Representative confocal microscopy images of γH2AX and 53BP1 staining in control NTC and Loxl3-silenced (sh2 and sh3) MeL3 cells. Nuclei were counterstained with DAPI (blue). Scale bars: 20 µm. (<b>H</b>) Quantification of γH2AX and 53BP1 foci per nucleus in cells shown in (<b>G</b>). A total of 100–150 cells were analyzed per condition (NTC, sh2, and sh3). Mean ± SEM of <span class="html-italic">n</span> = 3–5 independent experiments is depicted (<b>E</b>,<b>F</b>,<b>H</b>). The <span class="html-italic">p</span>-values were calculated by two-sided unpaired Student’s <span class="html-italic">t</span>-test (<b>E</b>,<b>F</b>,<b>H</b>). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 5
<p>Loxl3 favors melanoma progression. (<b>A</b>) Results from three independent tumorigenesis assays of orthotopically injected control (NTC) and Loxl3-silenced (sh2 and sh3) MeL3 cells in immunodeficient mice. The chart (left) includes the tumor incidence at 21 days after injection, average tumor latency, and minimal to maximal latency of <span class="html-italic">n</span> = 10 tumors per condition. The graph (right) depicts tumor volume growth up until 21 days after cell injection. (<b>B</b>) Volume of tumors originated by control (NTC) and Loxl3-depleted MeL3 cells measured at endpoint (<span class="html-italic">n</span> = 10 tumors per condition). (<b>C</b>) Number of migrated control (NTC) and Loxl3-silenced (sh2 and sh3) MeL3 cells detected 24 h after seeding. Mean ± SEM of three independent experiments is shown (<b>A</b>–<b>C</b>). The <span class="html-italic">p</span>-values were calculated by two-sided unpaired Student´s <span class="html-italic">t</span>-test. ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 6
<p>Loxl3 is involved in melanoma metastasis. (<b>A</b>) Western blot showing Loxl3 silencing in B16-F10 cell line. Gapdh was used as a loading control. Uncropped Western Blots and densitometry analyses can be found at <a href="#app1-cancers-14-01200" class="html-app">supplementary original figures</a>. (<b>B</b>) Cell proliferation analyses of control (NTC) and Loxl3-silenced (sh2 and sh3) B16-F10 cells at indicated time points after seeding represented as % of cell growth. Mean ± SEM of <span class="html-italic">n</span> = 3 independent experiments is depicted. (<b>C</b>) Tumorigenesis assays of orthotopically injected control (NTC) and Loxl3-silenced (sh2 and sh3) B16-F10 cells. The graph (left) depicts tumor volume progression until endpoint (day 28 after cell injection). The chart includes tumor incidence (day 18), average tumor latency, and minimal to maximal latency (<span class="html-italic">n</span> = 6 tumors per condition). Mean ± SEM is depicted. (<b>D</b>) Representative confocal microscopy images of γH2AX and 53BP1 staining in control NTC and Loxl3-silenced (sh2 and sh3) B16-F10 cells. Nuclei were counterstained with DAPI (blue). Scale bars: 20 µm. (<b>E</b>) Quantification of γH2AX and 53BP1 foci per nucleus in cells shown in (<b>D</b>). A total of 100–150 cells were analyzed per condition (NTC, sh2, and sh3), and mean ± SEM of <span class="html-italic">n</span> = 3 independent experiments is depicted. (<b>F</b>) Representative images (top) and tissue H/E staining (bottom) of recovered lungs 15 days after intravenous injection of control NTC and Loxl3-silenced (sh2 and sh3) B16-F10 cells. Orange arrows point to melanocytic metastatic lesions. Scale bars: 500 µm. Blue frames indicate the amplified tissue area shown in the upper left quadrant inside the left and middle panel images. (<b>G</b>) Quantification of lung metastatic foci per mice (left) and total number of foci per condition (right) in recovered lungs from experimental metastasis assays detailed in (<b>F</b>). Mean ± SEM (<span class="html-italic">n</span> = 6 mice per condition) is included. The <span class="html-italic">p</span>-values were calculated by two-sided unpaired Student´s <span class="html-italic">t</span>-test (<b>B</b>,<b>C</b>,<b>E</b>,<b>G</b>). * <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001; ns: not significant.</p> "> Figure 7
<p>Loxl3 contributes to melanoma cell plasticity. (<b>A</b>) Gene expression analyses of <span class="html-italic">Loxl3</span>, <span class="html-italic">Mitf,</span> and indicated EMT-TFs in control (NTC) and Loxl3-silenced (sh2 and sh3) MeL3 cells relative to internal <span class="html-italic">Gapdh</span> mRNA levels and control cells (NTC). (<b>B</b>) Immunoblot detection of Loxl3; N-cadherin (Ncad); and Zeb1, Snail1, and Prrx1 EMT-TFs in control (NTC) and Loxl3-silenced (sh2 and sh3) MeL3 cells. Tubulin was used as a loading control. Uncropped Western Blots and densitometry analyses can be found at <a href="#app1-cancers-14-01200" class="html-app">supplementary original figures</a>. (<b>C</b>) Quantification of Loxl3, Snail1, and Prrx1 protein levels from blots depicted in (<b>B</b>) relative to control cells (NTC). Mean ± SEM of <span class="html-italic">n</span> = 4–5 independent experiments is shown. The <span class="html-italic">p</span>-values were calculated by two-sided unpaired Student´s <span class="html-italic">t</span>-test (<b>A</b>,<b>C</b>). * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001; ns: not significant. (<b>D</b>) <span class="html-italic">PRRX1</span> and <span class="html-italic">LOXL3</span> gene expression analyses in a cohort of human melanoma cell lines relative to <span class="html-italic">L32</span> mRNA levels. (<b>E</b>) Immunoblot analyses of LOXL3, SNAIL1, and PRRX1 in human melanoma cell lines. Cell lines are grouped based on <span class="html-italic">NRAS<sup>Q61L</sup></span>- or <span class="html-italic">BRAF<sup>V600E</sup></span>-prevalent mutations. WT stands for triple WT subtype, which comprises melanomas lacking hot-spot mutations in <span class="html-italic">BRAF</span>, <span class="html-italic">RAS,</span> or <span class="html-italic">NF1</span> genes. Uncropped Western Blots and densitometry analyses can be found at <a href="#app1-cancers-14-01200" class="html-app">supplementary original figures</a>. (<b>F</b>) Scatter plots of <span class="html-italic">LOXL3</span> versus <span class="html-italic">SNAI1</span> (left) or <span class="html-italic">PRRX1</span> (middle) or <span class="html-italic">PRRX1</span> versus <span class="html-italic">SNAI1</span> (right) expression for samples in the skin cutaneous melanoma (SKCM) cohort from the TCGA (<span class="html-italic">n</span> = 469). The red solid line in each figure represents the regression line. Pearson correlation coefficient (R) with significance (<span class="html-italic">p</span>-value) is depicted in each panel. (<b>G</b>) Boxplots showing <span class="html-italic">SNAI1</span> or <span class="html-italic">PRRX1</span> expression plotted against normalized <span class="html-italic">LOXL3</span> mRNA levels in human melanoma samples from the TCGA (high expression <span class="html-italic">n</span> = 287, low expression <span class="html-italic">n</span> = 182). (<b>H</b>) Boxplots showing <span class="html-italic">LOXL3</span>, <span class="html-italic">SNAI1,</span> and <span class="html-italic">PRRX1</span> mRNA levels according to <span class="html-italic">BRAF</span> mutational status in melanoma samples from the TCGA cohort (<span class="html-italic">n</span> = 469).</p> ">
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Melanoma Mouse Model and Genotyping
2.2. Tamoxifen Treatment
2.3. Tumorigenesis Assays
2.4. Metastasis Assays
2.5. Histology and Immunohistochemistry
2.6. Primary Cell Cultures
2.7. Human Melanoma Cell Culture
2.8. Mouse Melanoma Cell Lines
2.9. Loxl3 Interference
2.10. Cell Proliferation Assays
2.11. Migration Assays
2.12. Annexin V Staining
2.13. Preparation of Cell Protein Extracts and Immunoblot Analyses
2.14. Immunofluorescence
2.15. DNA Foci Quantification
2.16. RNA Isolation, Reverse Transcription, and qPCR Analysis
2.17. Human Melanoma Data Mining and Analysis
2.18. Statistical Analyses
3. Results
3.1. Generation and Characterization of a Loxl3 Conditional Melanoma Mouse Model
3.2. Deletion of Loxl3 Increases Latency and Reduces Melanoma Tumor Growth and Overall Mice Survival
3.3. Selective Inactivation of Loxl3 in the Melanocytes Decreases Metastatic Dissemination
3.4. Loxl3 Silencing Is Detrimental to Melanoma Cell Growth
3.5. Loxl3 Expression in Melanoma Cells Contributes to Tumor Progression
3.6. Loxl3 Is Involved in Melanoma Cell Plasticity
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Vázquez-Naharro, A.; Bustos-Tauler, J.; Floristán, A.; Yuste, L.; Oltra, S.S.; Vinyals, A.; Moreno-Bueno, G.; Fabra, À.; Portillo, F.; Cano, A.; et al. Loxl3 Promotes Melanoma Progression and Dissemination Influencing Cell Plasticity and Survival. Cancers 2022, 14, 1200. https://doi.org/10.3390/cancers14051200
Vázquez-Naharro A, Bustos-Tauler J, Floristán A, Yuste L, Oltra SS, Vinyals A, Moreno-Bueno G, Fabra À, Portillo F, Cano A, et al. Loxl3 Promotes Melanoma Progression and Dissemination Influencing Cell Plasticity and Survival. Cancers. 2022; 14(5):1200. https://doi.org/10.3390/cancers14051200
Chicago/Turabian StyleVázquez-Naharro, Alberto, José Bustos-Tauler, Alfredo Floristán, Lourdes Yuste, Sara S. Oltra, Antònia Vinyals, Gema Moreno-Bueno, Àngels Fabra, Francisco Portillo, Amparo Cano, and et al. 2022. "Loxl3 Promotes Melanoma Progression and Dissemination Influencing Cell Plasticity and Survival" Cancers 14, no. 5: 1200. https://doi.org/10.3390/cancers14051200