MicroRNA-200 promotes lung cancer cell growth through FOG2-independent AKT activation

Abstract

MicroRNA-200 (miR-200) has emerged as a regulator of the PI3K/AKT pathway and cancer cell growth. It was reported that miR-200 can activate PI3K/AKT by targeting FOG2 (friend of GATA 2), which directly binds to the p85α regulatory subunit of PI3K. We found that miR-200 was elevated in early stage lung adenocarcinomas when compared with normal lung tissues, and the expression of miR-200 promoted the tumor spheroid growth of lung adenocarcinoma cells. We show that AKT activation was essential for such oncogenic action of miR-200. However, depletion of FOG2 had little effect on AKT activation. By performing a reverse-phase protein array, we found that miR-200 not only activated AKT but also concomitantly inactivated S6K and increased IRS-1, an S6K substrate that is increased on S6K inactivation. Depletion of IRS-1 partially inhibited the miR-200-dependent AKT activation. Taken together, our results suggest that miR-200 may activate AKT in lung adenocarcinoma cells through a FOG2-independent mechanism involving IRS-1. Our findings also provide evidence that increased miR-200 expression may contribute to early lung tumorigenesis and that AKT inhibitors may be useful for the treatment of miR-200-dependent tumor cell growth.

Keywords: AKT; lung cancer; microRNA-200; signaling.

Figure 1

miR-200 promotes lung adenocarcinoma cell growth. (A and B) The expression levels of miR-200a (A) and miR-200c (B) were quantitated by qPCR and normalized to that of RNU24 (internal control for small non-coding RNAs). (C) Light microscopic images of tumor spheroids formed by 344SQ lung adenocarcinoma cells that stably express either an empty vector or miR-200b/a/429.

Figure 2

miR-200 and ZEB1 reversely regulate key signaling components of the PI3K/AKT pathway. (A and B) Western blotting of pAKT (pS473), AKT, pp70S6K (pT389), p70S6K, pS6 (S235/S236), and S6 for 344SQ lung adenocarcinoma cells that express either an empty vector or a miR-200b/a/429 plasmid (A) and 393P lung adenocarcinoma cells that express either an empty vector or ZEB1 (B). (C) Tumor spheroids formed by 344SQ lung adenocarcinoma cells expressing either an empty vector or miR-200b/a/429 were treated with DMSO, GSK2141795 (2 μM), and rapamycin (500 nM). Tumor spheroids were counted and expressed as means ± S.D. * indicates that the t-test’s p value is less than 0.05 (statistically significant).

Figure 3

FOG2 is not required for AKT activation. (A) Western blotting of FOG2 and Actin (as a loading control) for 393Pzeb1 lung adenocarcinoma cells. (B) Western blotting of FOG2, pAKT (pS473), and AKT for 344SQ (left panels) and HCC827zeb1 (right panels) lung adenocarcinoma cells that stably express either non-targeting shRNA (N.T. shRNA) or FOG2 specific shRNAs. (C) Knockdown of FOG2 had no effect on 344SQ soft agar colony growth.

Figure 4

IRS-1 is partially involved in miR-200-dependent AKT activation. (A) RPPA array for 344SQ lung adenocarcinoma cells that stably express either an empty vector or miR-200b/a/429 plasmid. * and ** indicate that the t-test’s p values are less than 0.05 or 0.01, respectively (statistically significant or very significant). (B) Western blotting of IRS-1, pAKT (pS473), AKT and Actin for 344SQ lung adenocarcinoma cells expressing an empty vector or miR-200b/a/429 that transfected with control non-targeting siRNA (N.T. siRNA) or IRS-1 siRNA. (C) Western blotting of p85α, pAKT (pS473), and Actin showing knockdown of p85α suppresses AKT phosphorylation.

Figure 5

Schematic for the mechanism of miR-200-mediated AKT activation and tumor cell growth.