Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>Fascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC.</p> <p>Methods</p> <p>To understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients.</p> <p>Results</p> <p>Fascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (<it>P </it>= 0.041), increased lymph node metastasis (<it>P </it>= 0.001), less differentiation (<it>P </it>= 0.005), increased recurrence (<it>P </it>= 0.038) and shorter survival (<it>P </it>= 0.004) of the patients.</p> <p>Conclusion</p> <p>In conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.</p

Understanding the Role of Keratins 8 and 18 in Neoplastic Potential of Breast Cancer Derived Cell Lines

<div><h3>Background</h3><p>Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood.</p> <h3>Methodology/Principal Findings</h3><p>In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, <em>in-vitro</em> invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion <em>in-vitro</em>, while MCF10A did not show any changes in cell transformation assays.</p> <h3>Conclusions/Significance</h3><p>These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.</p> </div

Analysis of change in motility on K8 up−/down-regulation.

<p>Representative 10X Phase contrast images of time lapse microscopy at 0 hour and 20 hours showing wound healing (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing % wound closure at the end of 20 hours of MDA MB 435 K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>B</b>) MDAMB 468 K8 down-regulated (ShC1) and vector control (Vc) clone. Histogram showing % wound closure at the end of 20 hours of MDA MB 468 K8 down-regulated (ShC1, C2and C3) and vector control (Vc) clones (lower panel). (<b>C</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clone. Histogram showing % wound closure at the end of 20 hours of MCF10A K8 down-regulated (MShC1, C2and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed. Migration rate was calculated by AxioVision software. <b>Note:</b> Significant Increase in motility in K8 down-regulated MDA MB 468 clones as compared to vector control. Motility of K8 up−/down-regulated clones by transwell assay: Representative images of H &E stained migrated cells (<b>D</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of migrated cells at the end of 16 hours of MDA MB 435K8 over-expressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>E</b>) MDAMB 468 K8 down-regulated (ShC1) and vector control (Vc) clone. Histogram showing number of migrated cells at the end of 16 hours of MDA MB 468 K8 knockdown clones (ShC1, C2 and C3) and vector control (Vc) clones (lower panel). (<b>F</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clone. Histogram showing number of migrated cells of MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed <b>Note:</b> Significant decrease in motility in K8 over-expressed MDA MB 435 clones and significant increase in motility in K8 down-regulated MDA MB 468 clones (**p<0.01 by students t-test).</p

Analysis of change in <i>in-vitro</i> invasion on K8 up−/down-regulation.

<p>Representative 10X images of H&E stained membrane showing invaded cells. (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of invaded cells of MDA MB 435 K8 overexpressed (K8C1 and C2) and vector control (K8Vc) clones (lower panel). (<b>B</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing number of invaded cells of MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones (lower panel). (<b>C</b>) K8 down-regulated MCF10A (MShC1) and vector control (MVc) clones. Histogram showing number of invaded cells of MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones (lower panel). Results are mean of ± SE of three independent experiments performed <b>Note:</b> Decreased invasion in K8 over-expressed MDA MB 435 clones (K8C1 and C2) and increased invasion in K8 down-regulated MDA MB 468 clones (ShC1, C2, and C3) as compared to their respective vector controls (K8Vc) and (Vc).</p

Analysis of vimentin expression in K8 (over-expressed) MDA-MB-435 and K8 (down-regulated) MDA-MB-468 and MCF10A clones.

<p>Representative confocal images of vimentin filaments (<b>A</b>) K8 over-expressed MDA MB 435 (K8C1) and vector control (K8Vc) clones. (<b>B</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. (<b>C</b>) MCF10A K8 down-regulated (MshC1) and vector control (MVc) clones. Scale bar: 10 µm. For measuring the intensity of the cells the 4% laser with emission filter band pass of 505–550 was used. All the scanning conditions of gain offset and laser percentage were kept same and applied for all images of vector and their respective clones with secondary control as threshold. The mean fluorescence intensity of 20 cells was calculated and the average of three independent experiments was calculated. Results are mean of ± SE of three independent experiments performed <b>Note:</b> No change in vimentin expression or filament formation on K8 up−/down-regulation.</p

Analysis of changes in soft agar colony forming potential and cell proliferation on K8 up−/down-regulation.

<p>Representative phase contrast images (10X) of colonies formed in soft agar (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1) and vector control (K8Vc) clones. Histogram showing number of colonies of MDA MB 435 K8 overexpressed (K8C1, and C2) and vector control (K8Vc) clones (right hand side). (<b>C</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. Histogram showing number of colonies of MDA MB 468 K8 down-regulated (ShC1, C2and C3) and vector control (Vc) clones (right hand side). (<b>E</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clones. Histogram showing number of colonies of MCF10A K8 down-regulated (MshC1, C2 and C3) and vector control (MVc) clones (right hand side). <b>Note:</b> Increase in soft agar colonies formed in K8 down-regulated MDA MB 468 clones (*p<0.05 by students t-test). Cell proliferation curves of (<b>B</b>) MDA MB 435 K8 overexpressed (K8C1, C2 and C3) and vector control (K8Vc) clones. (<b>D</b>) MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. (<b>F</b>) MCF10A K8 down-regulated (MShC1, C2 and C3) and vector control (MVc) clones. Cell proliferation was plotted against time. Results are mean ± SE of three independent experiments performed in triplicate. <b>Note:</b> Decrease in proliferation in K8 over-expressed clones of MDA MB 435 (*p<0.05, **p<0.01 by students t-test).</p

Analysis of K8 and K18 expression in K8 (over-expressed) MDA MB 435 and K8 (down-regulated) MDA MB 468 and MCF10A clones.

<p>Western blot analysis of K8/18 using mAbs to K8 and K18 respectively (<b>A</b>) MDA MB 435 K8 over-expressed (K8C1, C2 and C3) and vector control (K8Vc) clones. (<b>C</b>) MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. (<b>E</b>) MCF10A K8 down-regulated (MshC1, C2 and C3) and vector control (MVc) clones. β-actin was taken as loading control. Representative confocal images of K8 and K18 filaments (<b>B</b>) K8 over-expressed in MDA MB 435 (K8C1) and vector control (K8Vc) clones. (<b>D</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. (<b>F</b>) MCF10A K8 down-regulated (MShC1) and vector control (MVc) clones. Scale bar: 10 µm. <b>Note:</b> Formation of K18 filaments in K8 over-expressed MDAMB 435 clones; No change in K18 filaments in K8 down-regulated MDA MB 468 clones; diffused K18 filament formation in K8 down-regulated MCF10A clones.</p

Analysis of change in tumorigenicity and metastatic potential on K8 up−/down- regulated clones of MDA MB 435 and MCF10A cell lines.

<p>(<b>A</b>) Representative images of NOD-SCID mice bearing tumors of vector control (K8Vc) and K8 over-expressed (K8C1) clones, 7 weeks after the injection in mammary fat pad. (<b>B</b>) Tumor growth was plotted against time (*p<0.05, **p<0.01 by student’s t-test). Results are mean of ± SE for five animals injected for each clone. (<b>C</b>) Representative images of excised lungs of animals injected with vector control (K8Vc) and K8 over-expressed (K8C1) clones. (<b>D</b>) H&E stained lung sections of animals injected with MDA MB 435 vector control clone (K8Vc) showing metastatic foci throughout the lungs and K8 over-expressed clone (K8C1) showing no metastasis. (<b>E</b>) Representative images of NOD-SCID mice injected with MCF10A vector control (MVc) and K8 down-regulated (MShC1) clones, 7 weeks after the injection in mammary fat pad. <b>Note:</b> Lungs of animals injected with MDA MB 435 vector control clone showing metastatic nodules and lungs of animals injected with K8 over-expressed MDAMB 435 clone (K8C1) showing no visible metastatic nodules.</p

Analysis of E-cadherin expression and β4 expression on K8 up−/down-regulation.

<p>E-cadherin expression: Representative confocal images of E-cadherin in (<b>A</b>) K8 over-expressed MDA MB 435 (K8C1) and vector control (K8Vc) clones. (<b>C</b>) MDA MB 468 K8 down-regulated (ShC1) and vector control (Vc) clones. (<b>E</b>) MCF10A K8 down-regulated (MC1) and vector control (MVc) clones. Scale bar: 10 µm. Note: Cytoplasmic localization of E-cadherin in K8 down-regulated MDA MB 468 clones, Nuclei (blue) is stained with DAPI. Western blot analysis of E-cadherin: (<b>B</b>) MDA MB 435 K8 over-expressed (K8C1, C2 and C3) and vector control (K8Vc) clones. MCF7 served as positive control. (<b>D</b>) MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. (<b>F</b>) MCF10A K8 down-regulated (MshC1, C2 and C3) and vector control (MVc) clones. β-actin was used as loading control. <b>Note:</b> No change in E-cadherin levels on K8 up−/down-regulation. (<b>G</b>) Western blot analysis of β4 integrin: in MDA MB 435 K8 over-expressed (K8C1, C2 and C3) and vector control (K8Vc) clones and MDA MB 468 K8 down-regulated (ShC1, C2 and C3) and vector control (Vc) clones. β-actin was taken as loading control. <b>Note:</b> No change in levels of β4 integrin.</p