MEN1 silencing triggers the dysregulation of mTORC1 and MYC pathways in ER+ breast cancer cells.

Menin, encoded by the MEN1 gene, has been identified as a critical factor regulating ESR1 transcription, playing an oncogenic role in ER+ breast cancer (BC) cells. Here, we further dissected the consequences of menin inactivation in ER+ BC cells by focusing on factors within two major pathways involved in BC, mTOR and MYC. MEN1 silencing in MCF7 and T-47D resulted in an increase in phosphor-p70S6K1, phosphor-p85S6K1 and phosphor-4EBP1 expression. The use of an AKT inhibitor inhibited the activation of S6K1 and S6RP triggered by MEN1 knockdown (KD). Moreover, MEN1 silencing in ER+ BC cells led to increased formation of the eIF4E and 4G complex. Clinical studies showed that patients with menin-low breast cancer receiving tamoxifen plus everolimus displayed a trend toward better overall survival. Importantly, MEN1 KD in MCF7 and T-47D cells led to reduced MYC expression. ChIP analysis demonstrated that menin bound not only to the MYC promoter but also to its 5' enhancer. Furthermore, E2-treated MEN1 KD MCF7 cells displayed a decrease in MYC activation, suggesting its role in estrogen-mediated MYC transcription. Finally, expression data mining in tumors revealed a correlation between the expression of MEN1 mRNA and that of several mTORC1 components and targets and a significant inverse correlation between MEN1 and two MYC inhibitory factors, MYCBP2 and MYCT1, in ER+ BC. The current work thus highlights altered mTORC1 and MYC pathways after menin inactivation in ER+ BC cells, providing insight into the crosstalk between menin, mTORC1 and MYC in ER+ BC.

Reduced menin expression leads to decreased ERα expression and is correlated with the occurrence of human luminal B-like and ER-negative breast cancer subtypes.

PURPOSE: Menin, encoded by the MEN1 gene, was recently reported to be involved in breast cancers, though the underlying mechanisms remain elusive. In the current study, we sought to further determine its role in mammary cells. METHODS: Menin expression in mammary lesions from mammary-specific Men1 mutant mice was detected using immunofluorescence staining. RT-qPCR and western blot were performed to determine the role of menin in ERα expression in human breast cancer cell lines. ChIP-qPCR and reporter gene assays were carried out to dissect the action of menin on the proximal ESR1 promoter. Menin expression in female patients with breast cancer was analyzed and its correlation with breast cancer subtypes was investigated. RESULTS: Immunofluorescence staining revealed that early mammary neoplasia in Men1 mutant mice displayed weak ERα expression. Furthermore, MEN1 silencing led to both reduced ESR1 mRNA and ERα protein expression in MCF7 and T47D cells. To further dissect the regulation of ESR1 transcription by menin, we examined whether and in which way menin could regulate the proximal ESR1 promoter, which has not been fully explored. Using ChIP analysis and reporter gene assays covering - 2500 bp to + 2000 bp of the TSS position, we showed that the activity of the proximal ESR1 promoter was markedly reduced upon menin downregulation independently of H3K4me3 status. Importantly, by analyzing the expression of menin in 354 human breast cancers, we found that a lower expression was associated with ER-negative breast cancer (P = 0.041). Moreover, among the 294 ER-positive breast cancer samples, reduced menin expression was not only associated with larger tumors (P = 0.01) and higher SBR grades (P = 0.005) but also with the luminal B-like breast cancer subtype (P = 0.006). Consistent with our clinical data, we demonstrated that GATA3 and FOXA1, co-factors in ESR1 regulation, interact physically with menin in MCF7 cells, and MEN1 knockdown led to altered protein expression of GATA3, the latter being a known marker of the luminal A subtype, in MCF7 cells. CONCLUSION: Taken together, our data provide clues to the important role of menin in ERα regulation and the formation of breast cancer subtypes.

Men1 disruption in Nkx3.1-deficient mice results in ARlow/CD44+ microinvasive carcinoma development with the dysregulated AR pathway.

Dysregulated androgen receptor (AR) plays a crucial role in prostate cancer (PCa) development, though further factors involved in its regulation remain to be identified. Recently, paradoxical results were reported on the implication of the MEN1 gene in PCa. To dissect its role in prostate luminal cells, we generated a mouse model with inducible Men1 disruption in Nkx3.1-deficient mice in which mouse prostatic intraepithelial neoplasia (mPIN) occur. Prostate glands from mutant and control mice were analyzed pathologically and molecularly; cellular and molecular analyses were carried out in PCa cell lines after MEN1 knockdown (KD) by siRNA. Double-mutant mice developed accelerated mPIN and later displayed microinvasive adenocarcinoma. Markedly, early-stage lesions exhibited a decreased expression of AR and its target genes, accompanied by reduced CK18 and E-cadherin expression, suggesting a shift from a luminal to a dedifferentiated epithelial phenotype. Intriguingly, over 60% of menin-deficient cells expressed CD44 at a later stage. Furthermore, MEN1 KD led to the increase in CD44 expression in PC3 cells re-expressing AR. Menin bound to the proximal AR promoter and regulated AR transcription via the H3K4me3 histone mark. Interestingly, the cell proliferation of AR-dependent cells (LNCaP, 22Rv1, and VCaP), but not of AR-independent cells (DU145, PC3), responded strongly to MEN1 silencing. Finally, menin expression was found reduced in some human PCa. These findings highlight the regulation of the AR promoter by menin and the crosstalk between menin and the AR pathway. Our data could be useful for better understanding the increasingly reported AR-negative/NE-negative subtype of PCa and the mechanisms underlying its development.