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. 2015 Nov;139(11):1349-61.
doi: 10.5858/arpa.2014-0471-OA. Epub 2015 Jul 14.

MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma

Lizandra Jimenez, Ved P Sharma, John Condeelis, Thomas Harris, Thomas J Ow, Michael B Prystowsky, Geoffrey Childs, Jeffrey E Segall  1
Affiliations

MicroRNA-375 Suppresses Extracellular Matrix Degradation and Invadopodial Activity in Head and Neck Squamous Cell Carcinoma

Lizandra Jimenez et al. Arch Pathol Lab Med. 2015 Nov.

Abstract

Context: Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated degradation of the extracellular matrix.

Objective: To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity.

Design: For evaluation of the matrix degradation properties of the HNSCC lines, an invadopodial matrix degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases.

Results: Increased miR-375 expression in HNSCC cells suppresses extracellular matrix degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases.

Conclusions: MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.

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Figures

Figure 1
Figure 1
Increased microRNA-375 expression impairs extracellular matrix degradation and number of mature invadopodia in UMSCC1 cells. Representative images of fluorescent matrix (A, D), tyrosine kinase substrate with 5 SH3 domains (Tks5) (B, E), and cortactin (C, F) immunostaining for UMSCC1 transductant cells. Arrows in zoomed areas show matrix degradation holes colocalized with Tks5 and cortactin puncta. Quantitation of matrix degradation per cell for UMSCC1 transductant cells (G) shown as mean and standard error of the mean (SEM) of 4 independent experiments; **P=.002. Quantitation of invadopodium precursors and mature invadopodia per cell for UMSCC1 transductant cells (H) shown as mean and SEM of 4 independent experiments; *P=.02 (original magnification × 413 [A through F]; original magnification × 1270 [insets A through F]). Abbreviation: n.s., not significant.
Figure 2
Figure 2
Increased microRNA-375 expression impairs extracellular matrix degradation and invadopodia numbers in OSC19 cells. Representative images of fluorescent matrix (A, D), tyrosine kinase substrate with 5 SH3 domains (Tks5) (B, E), and cortactin (C, F) immunostaining for OSC19 transductant cells. Arrows in zoomed areas show matrix degradation holes colocalized with Tks5 and cortactin puncta. Quantitation of matrix degradation per cell for OSC19 transductant cells (G) shown as mean and standard error of the mean (SEM) of 5 independent experiments; ***P< .001. Quantitation of invadopodium precursors and mature invadopodia per cell for OSC19 transductant cells (H) shown as mean and SEM of 5 independent experiments; *P=.01, ***P <.001 (original magnification ×413 [A through F]; original magnification ×1191 [insets A through F]).
Figure 3
Figure 3
Elevated microRNA-375 expression does not significantly alter total Tks5 (tyrosine kinase substrate with 5 SH3 domains), cortactin, and fascin protein levels in UMSCC1 cells. Representative Western blots of total Tks5 (A), cortactin (B), and fascin (C) protein levels in UMSCC1 transductant lines. β-Tubulin or β-actin was used as a loading control for Western blots. Quantitation of total Tks5, cortactin, and fascin levels in UMSCC1 transductant cells (D) shown as means and standard error of the mean of 5 independent experiments. Abbreviation: n.s., not significant.
Figure 4
Figure 4
Higher microRNA-375 expression does not reduce tyrosine phosphorylation of cortactin in UMSCC1 cells. A, Representative Western blots (WBs) for cortactin in total phosphotyrosine immunoprecipitates (IP pTYR) compared to the total cell lysate (5% input) and nonspecific rabbit immunoglobin G (IP Rabbit IgG) and beads (Beads only) control. B, Quantitation of cortactin levels in immunoprecipated phosphotyrosine proteins, shown as mean and standard error of the mean (SEM) of 4 independent experiments. C, Representative WBs for cortactin and phosphocortactin (pY421) in cortactin immunoprecipitates (IP Cortactin) compared to the total cell lysate (5% input) and nonspecific mouse IgG (IP Mouse IgG) and beads (Beads only) controls. D, Quantitation of phosphocortactin levels compared to total cortactin in cortactin immunoprecipitates, shown as mean and SEM of 4 independent experiments. Abbreviation: n.s., not significant.
Figure 5
Figure 5
Increased microRNA-375 expression in UMSCC1 cells did not diminish cortactin tyrosine phosphorylation at matrix degradation holes. Representative images of fluorescent matrix (A, D), cortactin (B, E), and phosphocortactin (pY421) (C, F) immunostaining for UMSCC1 transductant cells. Arrows in zoomed areas show matrix degradation holes colocalized with cortactin and phosphocortactin puncta. G, Quantitation of the ratio of signal intensity of total cortactin and phosphocortactin at matrix degradation holes, shown as mean and standard error of the mean of 3 independent experiments (original magnification ×413 [A through F]; original magnification ×953 [insets A through F]).
Figure 6
Figure 6
UMSCC1 cells expressing increased levels of microRNA-375 show dramatic reductions in protease secretion levels. Conditioned cell culture supernatants were harvested and assayed for protease secretion levels by using the Proteome Profiler Human Protease Arrays. A, Representative images of protease arrays incubated with cell culture supernatants from UMSCC1 transductant cells. B, Ratio of the 6 proteases that were most diminished in the protease array analyses, shown as means and standard error of the mean of 3 independent experiments; *P < .001. Abbreviations: ADAMTS-1, ADAM metal-lopeptidase with thrombospondin type 1 motif, 1; MMP-9, matrix metalloproteinase 9.
Figure 7
Figure 7
OSC19 cells expressing higher levels of microRNA-375 exhibit declines in secreted levels of proteases. Conditioned cell culture supernatants were harvested and assayed for protease secretion by using the Proteome Profiler Human Protease Arrays. A, Representative images of protease arrays incubated with cell culture supernatant from OSC19 transductant cells. B, Ratio of the 6 proteases that were most diminished in the protease array analyses, shown as means and standard error of the mean of 3 independent experiments; *P <.001. Abbreviation: MMP, matrix metalloproteinase.
Figure 8
Figure 8
Elevated expression of microRNA-375 in HNSCC cells significantly reduces kallikrein 6, kallikrein 10, and MMP-9 messenger RNA (mRNA) expression. The mRNA expression levels in the transductant lines were measured by TaqMan quantitative real-time polymerase chain reaction (qPCR). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control per cell line. Relative kallikrein 6 (KLK6), kallikrein 10 (KLK10), and matrix metal-loproteinase 9 (MMP9) mRNA expression levels in UMSCC1 and OSC19 transductant lines, shown in A through C, respectively. Data shown as mean and standard error of the mean of the fold change (2−ΔΔCT) from 5 independent experiments; ****P<.001.
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