The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth.

BACKGROUND: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors that can be treated with hormone therapy, or those with HER2-positive tumors that can be treated with anti-HER2 therapy. Therefore, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated the potential of a novel phosphatase, NUDT5, as a potential therapeutic target for TNBC. METHODS: The mRNA expression levels of NUDT5 in breast cancers were investigated using TCGA and METABRIC (Curtis) datasets. NUDT5 ablation was achieved through siRNA targeting and NUDT5 inhibition with the small molecule inhibitor TH5427. Xenograft TNBC animal models were employed to assess the effect of NUDT5 inhibition on in vivo tumor growth. Proliferation, death, and DNA replication assays were conducted to investigate the cellular biological effects of NUDT5 loss or inhibition. The accumulation of 8-oxo-guanine (8-oxoG) and the induction of γH2AX after NUDT5 loss was determined by immunofluorescence staining. The impact of NUDT5 loss on replication fork was assessed by measuring DNA fiber length. RESULTS: In this study, we demonstrated the significant role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. Our findings indicate that loss of NUDT5 results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is not attributed to cell death, but rather to the suppression of proliferation. The loss or inhibition of NUDT5 led to an increase in the oxidative DNA lesion 8-oxoG, and triggered the DNA damage response in the nucleus. The interference with DNA replication ultimately inhibited proliferation. CONCLUSIONS: NUDT5 plays a crucial role in preventing oxidative DNA damage in TNBC cells. The loss or inhibition of NUDT5 significantly suppresses the growth of TNBCs. These biological and mechanistic studies provide the groundwork for future research and the potential development of NUDT5 inhibitors as a promising therapeutic approach for TNBC patients.

Targeting the mTOR Pathway for the Prevention of ER-Negative Breast Cancer.

PREVENTION RELEVANCE: Our results show that everolimus delays mammary tumor formation in multiple mouse models, suggesting that mTOR inhibitors will be useful for the prevention of ER-negative and triple-negative breast cancer in humans. See related Spotlight, p. 787.

SOX9 Is Essential for Triple-Negative Breast Cancer Cell Survival and Metastasis.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial-mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. IMPLICATIONS: Our studies identified the SOX9 protein as a "master regulator" of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.

Mutant P53 induces MELK expression by release of wild-type P53-dependent suppression of FOXM1.

Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer, and is associated with a poor prognosis due to frequent distant metastasis and lack of effective targeted therapies. Previously, we identified maternal embryonic leucine zipper kinase (MELK) to be highly expressed in TNBCs as compared with ER-positive breast cancers. Here we determined the molecular mechanism by which MELK is overexpressed in TNBCs. Analysis of publicly available data sets revealed that MELK mRNA is elevated in p53-mutant breast cancers. Consistent with this observation, MELK protein levels are higher in p53-mutant vs. p53 wild-type breast cancer cells. Furthermore, inactivation of wild-type p53, by loss or mutation of the p53 gene, increases MELK expression, whereas overexpression of wild-type p53 in p53-null cells reduces MELK promoter activity and MELK expression. We further analyzed MELK expression in breast cancer data sets and compared that with known wild-type p53 target genes. This analysis revealed that MELK expression strongly correlates with genes known to be suppressed by wild-type p53. Promoter deletion studies identified a p53-responsive region within the MELK promoter that did not map to the p53 consensus response elements, but to a region containing a FOXM1-binding site. Consistent with this result, knockdown of FOXM1 reduced MELK expression in p53-mutant TNBC cells and expression of wild-type p53 reduced FOXM1 expression. ChIP assays demonstrated that expression of wild-type p53 reduces binding of E2F1 (a critical transcription factor controlling FOXM1 expression) to the FOXM1 promoter, thereby, reducing FOXM1 expression. These results show that wild-type p53 suppresses FOXM1 expression, and thus MELK expression, through indirect mechanisms. Overall, these studies demonstrate that wild-type p53 represses MELK expression by inhibiting E2F1A-dependent transcription of FOXM1 and that mutation-driven loss of wild-type p53, which frequently occurs in TNBCs, induces MELK expression by suppressing FOXM1 expression and activity in p53-mutant breast cancers.

The phosphatase PPM1A inhibits triple negative breast cancer growth by blocking cell cycle progression.

Estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative, or "triple negative," breast cancer (TNBC) is a poor prognosis clinical subtype that occurs more frequently in younger women and is commonly treated with toxic chemotherapy. Effective targeted therapy for TNBC is urgently needed. Our previous studies have identified several kinases critical for TNBC growth. Since phosphatases regulate the function of kinase signaling pathways, we sought to identify critical growth-regulatory phosphatases that are expressed differentially in ER-negative, as compared to ER-positive, breast cancers. In this study, we examined the role of one of these differentially expressed phosphatases, the protein phosphatase Mg + 2/Mn + 2 dependent 1A (PPM1A) which is underexpressed in ER-negative breast cancer as compared to ER-positive breast cancers, in regulating TNBC growth. We found that PPM1A is deleted in ~40% of ER-negative breast cancers, and that induced expression of PPM1A suppresses in vitro and in vivo growth of TNBC cells. This study demonstrates that induction of PPM1A expression blocks the cell cycle and reduces CDK and Rb phosphorylation. These results suggest PPM1A is a crucial regulator of cell cycle progression in triple negative breast cancer. Our results also suggest that PPM1A loss should be explored as a predictive biomarker of CDK inhibitor sensitivity.

Molecularly targeted therapies for p53-mutant cancers.

The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells.