Vishnubhotla Ramana, Sun Shan, Huq Jameela, Bulic Marinka, Ramesh Anil, Guzman Grace, Cho Michael, Glover Sarah C
Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA.
Lab Invest. 2007 Nov;87(11):1149-58. doi: 10.1038/labinvest.3700674. Epub 2007 Sep 17.
The ROCK-II isoform of Rho's downstream effector, Rho kinase, has been linked with greater invasion and metastasis in solid tumors. We have previously shown that ROCK-II is overexpressed at the advancing edge of colon cancers. The mechanism whereby ROCK-II contributes invasion, particularly in the setting of colon cancer, remains to be elucidated fully. To better understand its contribution, we evaluated ROCK-II expression in both non-malignant (NCM460 and IEC-6) and malignant (Caco-2 E, SW620, and HCT-116) intestinal epithelial cell lines grown in type I collagen scaffolds. Using multiphoton microscopy, we observed that ROCK-II localized to the actin cytoskeleton in non-malignant cells but localized to the cell periphery as focal collections with an absence of adjacent collagen in all colon cancer cell lines. By transmission electron microscopy, these collections corresponded with finger-like projections previously described as invadopodia. Immunogold staining with cortactin, matrix metalloprotease (MMP)-2, -9, and -13 confirmed that these were indeed invadopodia. To further link ROCK-II to colon cancer invasion, we treated non-malignant and malignant intestinal epithelial cell lines with ROCK-II siRNA and evaluated depth of invasion, proliferation, and MMP-2, -9, and -13 activities. The most striking effect was seen in the highly tumorigenic cell lines, SW620 and HCT-116, wherein ROCK-II knockdown resulted in a two-fold or more reduction in invasion. This reduction in invasion was not due to a decrease in cell proliferation, as a significant reduction in proliferation was only observed in the two non-malignant intestinal cell lines. Finally, both MMP-2 and -13 activities were significantly decreased in all colon cancer cell lines. Taken together, these data suggest for the first time that ROCK-II is a critical mediator of colon cancer cell invasion through its modulation of MMP-2 and -13 at the site of invadopodia but regulates proliferation in non-malignant intestinal cells.
Rho下游效应物Rho激酶的ROCK-II亚型与实体瘤中更强的侵袭和转移相关。我们之前已经表明，ROCK-II在结肠癌的前沿过度表达。ROCK-II促进侵袭的机制，尤其是在结肠癌的情况下，仍有待充分阐明。为了更好地理解其作用，我们评估了在I型胶原支架中生长的非恶性（NCM460和IEC-6）和恶性（Caco-2 E、SW620和HCT-116）肠上皮细胞系中ROCK-II的表达。使用多光子显微镜，我们观察到ROCK-II在非恶性细胞中定位于肌动蛋白细胞骨架，但在所有结肠癌细胞系中定位于细胞周边，形成焦点聚集，且没有相邻的胶原蛋白。通过透射电子显微镜，这些聚集物与先前描述为侵袭性伪足的指状突起相对应。用皮质肌动蛋白、基质金属蛋白酶（MMP）-2、-9和-13进行免疫金染色证实这些确实是侵袭性伪足。为了进一步将ROCK-II与结肠癌侵袭联系起来，我们用ROCK-II siRNA处理非恶性和恶性肠上皮细胞系，并评估侵袭深度、增殖以及MMP-2、-9和-13的活性。在高致瘤性细胞系SW620和HCT-116中观察到了最显著的效果，其中敲低ROCK-II导致侵袭减少两倍或更多。侵袭的减少不是由于细胞增殖的降低，因为仅在两个非恶性肠细胞系中观察到增殖有显著降低。最后，所有结肠癌细胞系中的MMP-2和-13活性均显著降低。综上所述，这些数据首次表明，ROCK-II通过在侵袭性伪足部位调节MMP-2和-13，是结肠癌细胞侵袭的关键介质，但在非恶性肠细胞中调节增殖。
