Co-targeting BET bromodomain BRD4 and RAC1 suppresses growth, stemness and tumorigenesis by disrupting the c-MYC-G9a-FTH1axis and downregulating HDAC1 in molecular subtypes of breast cancer

Abstract

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.

Keywords: BET bromodomain; BRD4; Breast cancer; FTH1; G9a; HDAC1; JQ1; NSC23766; RAC1; c-MYC.

Figure 1

Treatment with JQ1 or NSC inhibits different molecular subtypes of BRCA cell growth. MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were treated with increasing concentrations of JQ1 for 72 h and assessed for viability by (A) MTT assay and (B) crystal violet staining. (C) Mean ± SD of crystal violet absorbance values based on three independent experiments. MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were treated with increasing concentrations of NSC for 72 h and assessed for viability by (D) MTT assay and (E) crystal violet staining. (F) Mean ± SD of crystal violet absorbance values based on three independent experiments. Data presented is the mean ± SD of three independent experiments; *p < 0.05; **p < 0.005; ***p < 0.0005; # no significant difference.

Figure 2

Combined JQ1 plus NSC treatment suppresses growth and oncogenic potential of different molecular subtypes of BRCA cells. MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were treated with increasing concentrations of JQ1 and/or NSC for 72 h and assessed for viability by (A) MTT assay and (B) crystal violet staining. (C) Mean ± SD of crystal violet absorbance values based on three independent experiments. Data presented in A and C is the mean ±SD of three independent experiments; *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (D) MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were treated with JQ1 (0.5 uM) and/or NSC (15 uM) for 72 h and further grown in drug-free medium for 14-18 days; clonogenic potential was assessed using crystal violet staining. (E) Graph represents statistical analysis of clonogenic data. *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (F) SEM micrographs of MCF-7 cells treated with DMSO, JQ1 (2 uM) and/or NSC (30 uM) for 72 h and photographs were taken.

Figure 3

Combined JQ1 plus NSC treatment induces cellular senescence, inhibits mammosphere formation and cell migration in breast cancer cells. (A) MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were treated with JQ1 (0.5 uM) and/or NSC (15 uM) for 72 h and further grown in drug-free medium for 7 days to determine cellular senescence. (B) Statistical analysis of data from A; data presented is the mean ±SD of three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (C) MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were grown in ultra-low attachment plates and treated with JQ1 (0.5 uM) and/or NSC (15 uM) for 7 days to examine mammosphere formation. (D) Mean ±SD of mammosphere formation efficiency based on three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (E) MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells were treated with JQ1 (0.5 uM) and/or NSC (15 uM) for 72 h prior to performing the wound healing assay as means of determining cell migration potential. Wound healing area was measured at 24 h post scratching; images shown are representative of three independent experiments. (F) Histograms next to corresponding images is the mean ±SD of wound healing potential based on three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant.

Figure 4

NSC and JQ1 combined treatment suppresses MDA-MB-231 xenograft tumor growth. (A) Mice bearing MDA-MB-231-based tumors were treated with DMSO, NSC23776, JQ1 and combination three times per week for three weeks. (B) Representative images of tumor-bearing treated and control mice were taken on the concluding day (day 24) of the experiment. (C) Xenograft tumor growth curves of tumor-bearing control and treated mice; tumor size was measured at days 0, 3, 6, 9, 12, 15, 18, 21and 24. (D) Body-weight of tumor-bearing control and treated mice were taken at days 0, 3, 6, 9, 12, 15, 18, 21 and 24. (E) Final xenograft tumor weight was taken on day 24 of the experiment. Student t-test was performed for tumor volume and tumor weight statistical analysis. *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant.

Figure 5

Co-targeting BRD4 and RAC1 targets c-MYC-G9a-FTH1 axis and induces autophagy. The expression of c-MYC, G9a and FTH1 was examined in MCF-7, MDA-MB- 231, SKBR3 and JIMT-1 cells treated with (A) JQ1 (2 uM) and/or NSC (30 uM) or combination for 72 h; β-actin was used as a negative loading control. Expression status of (B) LC3BI/II was evaluated in MCF-7, MDA-MB-231, SKBR3 and/or JIMT-1 cells at 72 h following treatment with JQ1 (2 uM) and/or NSC (30 uM) for 72 h. β-actin was used as a negative loading control.

Figure 6

Effects of JQ1 and NSC treatment on the expression of key cellular signaling pathways and HDAC1/Ac-H3K9 axis. (A) The expression of BRD4, RAC1 and NFKB1 were examined in MCF-7, MDA-MB- 231, SKBR3 and JIMT-1 cells treated with JQ1 (2 µM) and/or NSC (30 µM) or combination for 72 h; β-actin was used as a negative loading control. (B) The expression of HDAC1 and AC-H3K9 were determined in MCF-7, MDA-MB- 231, SKBR3 and JIMT-1 cells treated with JQ1 (2 µM) and/or NSC (30 µM) or combination for 72 h; β-actin was used as a negative loading control.

Figure 7

Silencing of c-MYC further sensitizes BRCA cells to the anti-growth effects of JQ1 plus NSC treatment. Cell viability as determined by (A) MTT assay in MCF-7, MDA-MB-231 and JIMT-1 cells transfected with control siRNA or c-MYC siRNA and treated with JQ1 (0.5 uM) and/or NSC (15 uM) for 72 h. *p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (B) Knockdown efficiency of c-MYC in MCF-7, MDA-MB-231 and JIMT-1 cells transfected with control siRNA or c-MYC siRNA for 72 hr. (C) Mean ±SD of crystal violet absorbance values as calculated based on three independent experiments. p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (D) Crystal violet staining data in MCF-7, MDA-MB-231 and JIMT-1 cells transfected with control siRNA or c-MYC siRNA and treated with JQ1 (0.5 µM) and/or NSC (15 µM) for 72 h. (E) Mammosphere formation in MCF-7, MDA-MB-231 and JIMT-1 cultures transfected with control or c-MYC siRNA and treated with JQ1 (0.5 µM) and/or NSC (15 uM) for 7 days. Data is representative of three separate experiments. (F) Percentage mammosphere forming efficiency (%MFE ± SD) as calculated based on three separate experiments. p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (G) MDA-MB-231 and SKBR3 transfected with control or c-MYC siRNA and treated with JQ1 (0.5 µM) and/or NSC (15 µM) for 48 h prior to performing the wound healing assay. Wound healing area was measured at 24 h post scratching; images shown are representative of three independent experiments. (H) Quantification of wound healing data from G. Data represents mean ± SD of wound healing potential based on three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005; # no significant difference.

Figure 8

Vitamin C further sensitizes BRCA cells to the anti-growth effects of JQ1 plus NSC treatment. Cell viability as determined by (A) MTT assay or (B) Crystal violet staining in MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells treated with vitamin C (10 or 20 uM), JQ1 (0.5 uM) and NSC (15 uM) for 72 h. (C) Mean ±SD of crystal violet absorbance values as calculated based on three independent experiments. p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (D) Mammosphere formation in MCF-7, MDA-MB-231 and JIMT-1 cultures treated with vitamin C, JQ1 (0.5 uM) and NSC (15 uM) for 7 days. (E) Percentage mammosphere forming efficiency (%MFE ± SD) as calculated based on three separate experiments. p < 0.05; **p < 0.005; ***p < 0.0005; # not significant. (F) MDA-MB-231 and SKBR3 treated with vitamin C (10 or 20 µM) ± JQ1 (0.5 µM) and NSC (15 µM) for 48 h prior to performing the wound healing assay. Wound healing area was measured at 24 h post scratching; images shown are representative of three independent experiments. (G) Quantification of wound healing data from F. Data represents mean ± SD of wound healing potential based on three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005; # no significant difference.

Figure 9

Combined JQ1 plus NSC treatment does not promote ferroptosis in BRCA cells. (A) Expression of SLC7A11 and GPX4 proteins in MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells at 72 h -post-treatment with JQ1 (2 uM) and/or NSC (30 uM) for 72 h; β-actin was used as a negative loading control. (B) Cell viability as determined by crystal violet staining in MCF-7, MDA-MB-231, SKBR3 and JIMT-1 cells treated with Ferrostatin-1 (Fer-1) for 72 h in the presence/absence of JQ1 (2 uM) plus NSC (30 uM). (C) Mean ±SD of crystal violet absorbance values as calculated based on three independent experiments. *p < 0.05; **p < 0.005; ***p < 0.0005; # no significant difference.

Figure 10

Clinicopathalogical significance of RAC1 and BRD4 in BRCA. (A and B) Expression of RAC1 and BRD4 in basal-like, luminal-A, luminal-B and HER-2+ BRCA (PAM50 subtypes) molecular subtypes using the Breast Cancer Gene-Expression Miner v4.4 database. (C and D) Comparison of RAC1 and BRD4 expression in primary solid breast tumors vs normal solid breast tissue. Data was generated using DriverDBv3 database . (E and F) RAC1 and BRD4 expression profile in histological subtypes of breast cancer including Invasive ductal carcinoma (IDC), Invasive lobular breast cancer (ILC), mucinous breast cancer (Muc) and micropapillary breast cancer (Mic.). This data was generated using the Breast Cancer Gene-Expression Miner v4.4 database. (G-I) Survival analysis of RAC1 high and low expression in BRCA patients samples using prognoscan database. (J-L) Survival analysis of BRD4 high and low expression in BRCA patients samples using prognoscan database.

Figure 11

A hypothetical model illustrating: A) oncogenic signaling pathways promote BRD4 and RAC1 expressions and thereby activate oncogenic proteins, suppress tumor suppressor genes expression and target histone modification to facilitate cancer progression by targeting tumor growth, metastasis, cell migration, cancer stem cells (CSCs) expansion, drug resistance and autophagy. B) Combined inhibition of BRD4-RAC1 signaling pathways by BRD4 inhibitors (BRD4i) and RAC1 inhibitors (RAC1i) suppress c-MYC, G9a and HDAC1 oncogenic protein expressions and increase FTH1, LC3B and Ac-H3K9 levels to exert tumor suppressive effects. Combination of BRD4i and RACi coupled with c-MYC depletion or co-treatment of Vitamin C further suppress tumor growth, metastasis, cell migration, cancer stem cells (CSCs) expansion, drug resistance and induce autophagy.