N6-methyladenosine (m6A) modification is the most pervasive modification in mRNA, and has been considered as a new layer of epigenetic regulation on mRNA processing, stability and translation. Despite its functional significance in various physiological processes, the role of the m6A modification involved in breast cancer is yet fully understood. We used the m6A-RNA immunoprecipitation sequencing to identify the potential targets in breast cancer. To determine the underlying mechanism for the axis of FTO-BNIP3, we performed a series of in vitro and in vivo assays in 3 breast cancer cell lines and 36 primary breast tumor tissues and 12 adjunct tissues. We showed that FTO, a key m6A demethylase, was up-regulated in human breast cancer. High level of FTO was significantly associated with lower survival rates in patients with breast cancer. FTO promoted breast cancer cell proliferation, colony formation and metastasis in vitro and in vivo. We identified BNIP3, a pro-apoptosis gene, as a downstream target of FTO-mediated m6A modification. Epigenetically, FTO mediated m6A demethylation in the 3’UTR of BNIP3 mRNA and induced its degradation via an YTHDF2 independent mechanism. BNIP3 acts as a tumor suppressor and is negatively correlated with FTO expression in clinical breast cancer patients. BNIP3 dramatically alleviated FTO-dependent tumor growth retardation and metastasis. Our findings demonstrate the functional significance of the m6A modification in breast cancer, and suggest that FTO may serve as a novel potential therapeutic target for breast cancer.

N6‐methyladenosine (m ⁶ A) is an abundant nucleotide modification in mRNA, known to regulate mRNA stability, splicing, and translation, but it is unclear whether it is also has a physiological role in the intratumoral microenvironment and cancer drug resistance. Here, we find that METTL3, a primary m ⁶ A methyltransferase, is significantly down‐regulated in human sorafenib‐resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promotes sorafenib resistance and expression of angiogenesis genes in cultured HCC cells and activates autophagy‐associated pathways. Mechanistically, we have identified FOXO3 as a key downstream target of METTL3, with m ⁶ A modification of the FOXO3 mRNA 3′‐untranslated region increasing its stability through a YTHDF1‐dependent mechanism. Analysis of clinical samples furthermore showed that METTL3 and FOXO3 levels are tightly correlated in HCC patients. In mouse xenograft models, METTL3 depletion significantly enhances sorafenib resistance of HCC by abolishing the identified METTL3‐mediated FOXO3 mRNA stabilization, and overexpression of FOXO3 restores m ⁶ A‐dependent sorafenib sensitivity. Collectively, our work reveals a critical function for METTL3‐mediated m ⁶ A modification in the hypoxic tumor microenvironment and identifies FOXO3 as an important target of m ⁶ A modification in the resistance of HCC to sorafenib therapy.

Genomic deletion of the tumour suppressor TP53 frequently includes other neighbouring genes, such as the POLR2A housekeeping gene that encodes a crucial RNA polymerase II subunit; suppression of POLR2A with α-amanitin or by RNA interference selectively inhibits the tumorigenic potential of cancer cells, and in mouse models of cancer, tumours can be selectively targeted with α-amanitin coupled to antibodies, suggesting new therapeutic approaches for human cancers. The tumour suppressor gene TP53 is inactivated by mutation or deletion in a majority of human tumours. So far, attempts to restore the activity of its product, p53, have had little success owing to the complexity of p53 signalling. This paper suggests a new approach to targeting TP53 indirectly. Genomic deletion of TP53 frequently includes other neighbouring genes, such as the POLR2A housekeeping gene that encodes a crucial RNA polymerase II subunit. Xionbin Lu and colleagues show that loss of one copy of POLR2A renders cancer cells highly sensitive to inhibitors of RNA polymerase II, such as α-amanitin. In mouse models of cancer, tumours containing the POLR2A/TP53 co-deletion can be selectively targeted with α-amanitin conjugated to antibodies that target the cancer cells. Exploiting similar selective vulnerabilies for other genomic deletions that affect essential housekeeping in addition to tumour suppressor genes may pave a way towards selective therapies for a broad range of cancers. TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours1,2. A tremendous effort has been made to restore p53 activity in cancer therapies3,4,5,6,7. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin8,9. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity10. However, we found that α-amanitin-based antibody–drug conjugates are highly effective therapeutic agents with reduced toxicity11. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Chemotherapy for patients with metastatic colorectal cancer (CRC) is the standard of care, but ultimately nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may help identify novel targets and drugs that will, in turn, improve therapy. Oxaliplatin is a common component combination therapeutic regimen for use in patients with metastatic CRC, but is also used as a component of adjuvant therapy for patients at risk for recurrent disease. In this study, unbiased microRNA array screening revealed that the miR‐203 microRNA is up‐regulated in three of three oxaliplatin‐resistant CRC cell lines, and therefore we investigated the role of miR‐203 in chemoresistance. Exogenous expression of miR‐203 in chemo‐naïve CRC cells induced oxaliplatin resistance. Knockdown of miR‐203 sensitized chemoresistant CRC cells to oxaliplatin. In silico analysis identified ataxia telangiectasia mutated (ATM), a primary mediator of the DNA damage response, as a potential target of miR‐203. ATM mRNA and protein levels were significantly down‐regulated in CRC cells with acquired resistance to oxaliplatin. Using TCGA database, we identified a significant reverse correlation of miR‐203 and ATM expression in CRC tissues. We validated ATM as a bona fide target of miR‐203 in CRC cells. Mutation of the putative miR‐203 binding site in the 3′ untranslated region (3′UTR) of the ATM mRNA abolished the inhibitory effect of miR‐203 on ATM. Furthermore, stable knockdown of ATM induced resistance to oxaliplatin in chemo‐naïve CRC cells. This is the first report of oxaliplatin resistance in CRC cells induced by miR‐203‐mediated suppression of ATM.