Oncogenesis caused by loss of the SNF5 tumor suppressor is dependent on activity of BRG1, the ATPase of the SWI/SNF chromatin remodeling complex

Abstract

Alterations in chromatin play an important role in oncogenic transformation, although the underlying mechanisms are often poorly understood. The SWI/SNF complex contributes to epigenetic regulation by using the energy of ATP hydrolysis to remodel chromatin and thus regulate transcription of target genes. SNF5, a core subunit of the SWI/SNF complex, is a potent tumor suppressor that is specifically inactivated in several types of human cancer. However, the mechanism by which SNF5 mutation leads to cancer and the role of SNF5 within the SWI/SNF complex remain largely unknown. It has been hypothesized that oncogenesis in the absence of SNF5 occurs due to a loss of function of the SWI/SNF complex. Here, we show, however, distinct effects for inactivation of Snf5 and the ATPase subunit Brg1 in primary cells. Further, using both human cell lines and mouse models, we show that cancer formation in the absence of SNF5 does not result from SWI/SNF inactivation but rather that oncogenesis is dependent on continued presence of BRG1. Collectively, our results show that cancer formation in the absence of SNF5 is dependent on the activity of the residual BRG1-containing SWI/SNF complex. These findings suggest that, much like the concept of oncogene addiction, targeted inhibition of SWI/SNF ATPase activity may be an effective therapeutic approach for aggressive SNF5-deficient human tumors.

Figure 1. Inactivation of BRG1 and SNF5 have distinct effects upon cell survival and gene expression

(A) Immunoblots showing that Snf5 and Brg1 protein are efficiently ablated in Snf5^fl/fl or Brg1^fl/fl cells exposed to Cre recombinase. (B) Following treatment with Cre, proliferation of wild-type (WT), Snf5-deficient (Snf5^Δ/Δ), Brg1-deficient (Brg1^Δ/Δ), and Brg/Snf5 doubly-deficient cells (Snf5^Δ/Δ Brg1^Δ/Δ)were monitored. While cells lacking either Brg1 or Snf5 alone underwent growth arrest, combined loss of both Brg1 and Snf5 led to rapid cell death. (C) Primary MEFs can survive and proliferate in the absence of Brg1. MEFs isolated from three Brg1^fl/fl embryos (A, B, C) were exposed to a retrovirus expressing Cre-recombinase as in panel A and then maintained in selection. MEFs continue to stably proliferate in the absence of Brg1. To establish that MEFs continue to stably proliferate long-term in the absence of Brg1, MTT assays were performed on Brg1-deficinet and control MEFs. Representative proliferation data, from the day 65 to 78 time frame, is shown including serial re-plating on days 70 and 74. Immunoblots of Brg1 and actin at day 80 post infection is shown to confirm Brg1 absence. (D) Expression of Fmod reveals differences in Snf5 loss compared to Brg1 loss. Following Adeno-Cre treatment of conditional MEFs, real-time PCR reveals that Fmod expression is increased in Snf5 deficient MEFs but decreased in Brg1 deficient MEFs. Data represents level of expression of Fmod normalized to Actin message in three independent experiments ± S.E.

Figure 2. Brm can partially compensate for Brg1 loss in MEFs

(A) Brm protein levels modestly increase following the knockdown of Brg1 in MEFs. MEFs isolated from three wild-type (WT) and three Brg1^fl/fl embryos were exposed to a retro-virus expressing Cre-recombinase and under selection for 40 days. Immunoblots of Brm and Brg1 are shown. (B) MEFs isolated from three WT embryos were infected with either an RNAi-GFP-puro^r virus against both Brg1/Brm or a control GFP-puro^r virus. The following day, GFP+ cells were sorted by flow cytometry, and selected in puromycin for 2 days. Immunoblots show that the Brg1/BRM RNAi effectively suppresses Brg1 and Brm expression. Mouse brain was used as a positive staining control. (C) MEFs deficient for both Brg1 and Brm failed to proliferate and died. Graph represents the average of thee independent experiments ± S.E.

Figure 3. Co-inactivation of BRG1 and SNF5 affects cell survival and proliferation more severely than loss of either one alone

(A) Loss of Brg1 exacerbates the negative effects of Snf5 loss upon MEF survival. Graph represents the average survival ± S.E. from six independent experiments. The asterisk indicates statistical significance, p < 0.002. (B) Inactivation of Brg1 in T cells in vivo similarly exacerbates the effects of Snf5 loss. Splenocytes were isolated from CD4-Cre Brg1^fl/fl, CD4-Cre Snf5^fl/fl, and CD4-Cre Snf5^fl/fl, Brg1 ^fl/fl mice and CD3⁺ T cells were counted by flow cytometry. The graph represents the average number of cells ± S.E. from four mice of each genotype. The asterisk indicates statistical significance, p < 0.002.

Figure 4. Brg1, not Brm, is present and binds to other Swi/Snf components in Snf5 deficient tumors

(A) Western analysis of BRG1, BRM and SNF5 in human MRT cell lines. Hela cells were used as a positive control for expression. (B) Western analysis of Brg1, Brm and a Snf5 in Snf5-deficient murine lymphoma. CD8+ T cells and murine brain extracts were used as positive controls. Brg1 is present while Brm is not. (C) Brg1 binds to other Swi/Snf components in Snf5 deficient lymphomas. Whole cell extracts from murine Snf5 deficient lymphomas were subjected to Brg1 IP followed by immunoblotting. Jurkat cells were used as positive controls.

Figure 5. Knockdown of BRG1 is lethal to SNF5-deficient human tumor cell lines

(A) RNAi against BRG1 and BRM leads to stable knockdown of protein expression. Control SNF5 positive MDA321 breast adenocarcinoma cell lines or SNF5-deficient G401 MRT tumor cells lines were infected with a retrovirus containing an RNAi against BRG1/BRM or with a control retrovirus. Knock-down of BRG1 and BRM were confirmed by western following infection. BRM expression is not detectable in these cells (Figure 4). (B) CSF1 and SPARC remain dependent upon BRG1 for expression even in the absence of SNF5. Real-time PCR was used to detect CSF1 and SPARC gene expression following transduction of SNF5-deficient G401 cells with either BRG1/BRM or control RNAi. Data represents level of expression in presence of BRG1/BRM RNAi compared to control RNAi normalized to GADPH message in three independent experiments ± S.E. (C) SNF5 deficient MRT cells are dependent on the presence of BRG1 for cell growth. G401 MRT cells, or SNF5 positive SW13 and MDA231 cells were infected with either BRG1/BRM or control RNAi and monitored for proliferation. Data represent the means of four independent experiments ± SE. (D) Top panel: BrdU cell cycle analysis reveals that loss of Brg1 in SNF5-deficient G401 MRT cells, but not control MDA231 breast cancer cells, leads to decreased S phase (p = 2×10^-7) and G2/M (p = 0.001) phases accompanied by a increased G1 phase (p = 2×10^-6), indicating a G1 cell cycle arrest. Bottom panel: MRT cells die upon BRG1 knockdown. Photomicrographs following infection with either BRG1/BRM RNAi or control vector and seven days of selection.

Figure 6. Inactivation of Brg1 blocks tumor formation in Snf5-conditional mice

(A) Tumor-free survival curves of Lck-Cre Snf5^fl/fl (n=51), Lck-Cre Brg1^fl/fl (n=45), and Lck-Cre Snf5^fl/fl Brg1^fl/fl (n=9) mice. (B) Relative mRNA expression of Brg1 and Snf5 in CD8+ lymphomas obtained from Snf5^fl/fl or Snf5^fl/fl Brg1^fl/fl mice compared to expression in WT CD8+ T cells. While Snf5 expression is gone in the lymphomas, Brg1 is maintained. qPCR data is shown as the mean of two independent experiments ± S.E normalized to RPS8 control message. (C) Immunoblot showing that the lymphomas that ultimately arise in Snf5^fl/fl Brg1^fl/fl conditional mice are derived from cells in which Snf5 has been deleted but Brg1 retained. (D) A model showing that Cre induction in Lck-Cre or CD4-Cre Snf5^fl/fl Brg1^fl/fl mice can lead to four possible outcomes: 1) neither Snf5 nor Brg1 get excised, 2) both Snf5 and Brg1 get excised, 3) only Snf5, but not Brg1, gets excised, or 4) only Brg1, but not Snf5, gets excised following Cre induction. Our data reveals that lymphoma can only develop from the rare population of cells in which Snf5, but not Brg1, is excised and inactivated.