Previous studies have demonstrated the elevated expression of Rac1b in human breast and colorectal tumors. Similarly, we find that Rac1b is upregulated in a significant fraction of human lung adenocarcinomas examined. These findings suggest a tumor-promoting role for Rac1b in these cancers. Corroborating evidence to support this notion comes from in vitro studies in which the overexpression of Rac1b was shown to promote malignant transformation in a variety of cancer cell lines. In the current study, we focused on the tumor-promoting function of Rac1b in lung adenocarcinoma, in vivo. Towards this aim, we developed a mouse model with conditional activation of Rac1b expression. Using this model, we find that Rac1b alone is not sufficient to drive lung tumor initiation.
Analysis of K-ras status in the NSCLC lung adenocarcinomas indicated that activating K-ras mutations correlate significantly with elevated Rac1b expression. However, a significant number of tumors demonstrating elevated Rac1b expression were wild type for K-ras, suggesting that other mutations, perhaps in EGFR, might drive elevated Rac1b levels. Interestingly, previous studies in colorectal tumors demonstrate a correlation between B-Raf mutations and Rac1b upregulation, but no correlation with K-ras mutations (14
). These observed differences might be a reflection of the different tumor types examined.
Thus, while Rac1b overexpression alone did not promote tumorigenesis, the correlation with K-ras mutations suggests Rac1b could cooperate with K-ras activation. Indeed, by combining the Rosa26-LSL-Rac1b mice with a conditionally-activated oncogenic allele of K-ras, we find that Rac1b synergizes with oncogenic K-ras to facilitate lung cancer progression, in vivo. Comparative analysis of similar stage tumors from the different genotypes demonstrated no differences in rates of cell death, but did show a statistically significant increase in rates of cell proliferation in the tumors in LSL-K-rasG12D;Rosa26-LSL-Rac1b mice compared to the LSL-K-rasG12D mice. While the difference is small, over time it compounds to manifest as a significant increase in the tumor growth observed in vivo. Of note, our cell based studies in both human and mouse lung tumor cells that carry oncogenic K-ras mutations did not reveal a growth advantage when Rac1b is overexpressed. Given the subtle growth advantage conferred by Rac1b overexpression in vivo, the effects on proliferation in cultured cells might be below the level of detection possibly due to these cells being already at a maximal proliferative rate through the activity of other pathways. Further in vivo analysis will be required to unveil these mechanisms.
Our finding that Rac1b is not required for K-ras induced lung tumorigenesis stands in contrast to our previous findings demonstrating a requirement for Rac1 (11
). As an alternative splice variant of Rac1, Rac1b exhibits different features from Rac1. The extra 19 amino acid insertion in Rac1b accelerates GDP/GTP exchange and decreases hydrolysis of GTP. Rac1b is impaired in interaction with RhoGDI and the p21-activated kinases (PAKs) and fails to stimulate Rac1 signaling effectors such as c-Jun-NH2
-kinase and p38 mitogen-activated protein kinase (4
). Our studies in which we specifically knockdown either Rac1 or Rac1b demonstrate the specific requirement for Rac1 for cell proliferation. This suggests that signaling pathways regulated by Rac1, but not Rac1b, are likely to be required for K-ras induced transformation. Previous studies have indicated that Rac1b shows a diminished ability to activate the PAKs (4
). In support of this we find that overexpression of Rac1b did not enhance Pak1/2 activity. Likewise, in contrast to the knockdown of Rac1, the knockdown of Rac1b does not impact the activation state of the PAKs, which have been previously shown to be required for K-ras induced transformation in multiple tumor types (16
). Finally, recent studies have also suggested Rac1b can interact with downstream effectors that are not engaged by Rac1, such as p120 catenin (17
). Our findings therefore imply that these Rac1b –specific functions are not required for cell proliferation in the context of oncogenic K-ras.