A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation.

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

MCL-1 inhibition provides a new way to suppress breast cancer metastasis and increase sensitivity to dasatinib.

BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.

ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.

We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.

Short-term PI3K Inhibition Prevents Breast Cancer in Preclinical Models.

Antiestrogen medication is the only chemoprevention currently available for women at a high risk of developing breast cancer; however, antiestrogen therapy requires years to achieve efficacy and has adverse side effects. Therefore, it is important to develop an efficacious chemoprevention strategy that requires only a short course of treatment. PIK3CA is commonly activated in breast atypical hyperplasia, the known precancerous precursor of breast cancer. Targeting PI3K signaling in these precancerous lesions may offer a new strategy for chemoprevention. Here, we first established a mouse model that mimics the progression from precancerous lesions to breast cancer. Next, we demonstrated that a short-course prophylactic treatment with the clinically approved PI3K inhibitor alpelisib slowed early lesion expansion and prevented cancer formation in this model. Furthermore, we showed that alpelisib suppressed ex vivo expansion of patient-derived atypical hyperplasia. Together, these data indicate that the progression of precancerous breast lesions heavily depends on the PI3K signaling, and that prophylactic targeting of PI3K activity can prevent breast cancer. PREVENTION RELEVANCE: PI3K protein is abnormally high in breast precancerous lesions. This preclinical study demonstrates that the FDA-approved anti-PI3K inhibitor alpelisib can prevent breast cancer and thus warrant future clinical trials in high-risk women.

Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish.

Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models.

Tfap2b specifies an embryonic melanocyte stem cell that retains adult multifate potential.

Melanocytes, the pigment-producing cells, are replenished from multiple stem cell niches in adult tissue. Although pigmentation traits are known risk factors for melanoma, we know little about melanocyte stem cell (McSC) populations other than hair follicle McSCs and lack key lineage markers with which to identify McSCs and study their function. Here we find that Tfap2b and a select set of target genes specify an McSC population at the dorsal root ganglia in zebrafish. Functionally, Tfap2b is required for only a few late-stage embryonic melanocytes, and is essential for McSC-dependent melanocyte regeneration. Fate mapping data reveal that tfap2b+ McSCs have multifate potential, and are the cells of origin for large patches of adult melanocytes, two other pigment cell types (iridophores and xanthophores), and nerve-associated cells. Hence, Tfap2b confers McSC identity in early development, distinguishing McSCs from other neural crest and pigment cell lineages, and retains multifate potential in the adult zebrafish.

Targeting the Pro-survival Protein BCL-2 to Prevent Breast Cancer.

Current chemopreventive strategies require 3-5 years of continuous treatment and have the concerns of significant side effects; therefore, new chemopreventive agents that require shorter and safer treatments are urgently needed. In this study, we developed a new murine model of breast cancer that mimics human breast cancer initiation and is ideal for testing the efficacy of chemopreventive therapeutics. In this model, introduction of lentivirus carrying a PIK3CA gene mutant commonly found in breast cancers infects a small number of the mammary cells, leading to atypia first and then to ductal carcinomas that are positive for both estrogen receptor and progesterone receptor. Venetoclax is a BH3 mimetic that blocks the anti-apoptotic protein BCL-2 and has efficacy in treating breast cancer. We found that venetoclax treatment of atypia-bearing mice delayed the progression to tumors, improved overall survival, and reduced pulmonary metastasis. Therefore, prophylactic treatment to inhibit the pro-survival protein BCL-2 may provide an alternative to the currently available regimens in breast cancer prevention. PREVENTION RELEVANCE: This study demonstrates that prophylactic treatment with the BCL2-specific antagonist venetoclax prevents breast cancer initiated by a mutated and activated PIK3CA, the most common breast oncogene.

A Wnt-Independent LGR4-EGFR Signaling Axis in Cancer Metastasis.

Leucine-rich repeat-containing G protein-coupled receptors 4, 5, and 6 (LGR4/5/6) play critical roles in development and cancer. The widely accepted mechanism is that these proteins, together with their R-spondin ligands, stabilize Wnt receptors, thus potentiating Wnt signaling. Here we show that LGR4 enhanced breast cancer cell metastasis even when Wnt signaling was deactivated pharmacologically or genetically. Furthermore, LGR4 mutants that cannot potentiate Wnt signaling nevertheless promoted breast cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Multiomic screening identified EGFR as a crucial mediator of LGR4 activity in cancer progression. Mechanistically, LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a Wnt-independent LGR4-EGFR signaling axis with broad implications for cancer progression and targeted therapy. SIGNIFICANCE: This work demonstrates a Wnt-independent mechanism by which LGR4 promotes cancer metastasis.See related commentary by Stevens and Williams, p. 4397.

MCL-1 antagonism enhances the anti-invasive effects of dasatinib in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.

Myeloid cell leukemia 1 (MCL-1), an unexpected modulator of protein kinase signaling during invasion.

Myeloid cell leukemia-1 (MCL-1), closely related to B-cell lymphoma 2 (BCL-2), has a well-established role in cell survival and has emerged as an important target for cancer therapeutics. We have demonstrated that inhibiting MCL-1 is efficacious in suppressing tumour progression in pre-clinical models of breast cancer and revealed that in addition to its role in cell survival, MCL-1 modulated cellular invasion. Utilizing a MCL-1-specific genetic antagonist, we found two possible mechanisms; firstly MCL-1 directly binds to and alters the phosphorylation of the cytoskeletal remodeling protein, Cofilin, a protein important for cytoskeletal remodeling during invasion, and secondly MCL-1 modulates the levels SRC family kinases (SFKs) and their targets. These data provide evidence that MCL-1 activities are not limited to endpoints of extracellular and intracellular signaling culminating in cell survival as previously thought, but can directly modulate the output of SRC family kinases signaling during cellular invasion. Here we review the pleotropic roles of MCL-1 and discuss the implications of this newly discovered effect on protein kinase signaling for the development of cancer therapeutics.

The SKN-1/Nrf2 transcription factor can protect against oxidative stress and increase lifespan in C. elegans by distinct mechanisms.

In C. elegans, the skn-1 gene encodes a transcription factor that resembles mammalian Nrf2 and activates a detoxification response. skn-1 promotes resistance to oxidative stress (Oxr) and also increases lifespan, and it has been suggested that the former causes the latter, consistent with the theory that oxidative damage causes aging. Here, we report that effects of SKN-1 on Oxr and longevity can be dissociated. We also establish that skn-1 expression can be activated by the DAF-16/FoxO transcription factor, another central regulator of growth, metabolism, and aging. Notably, skn-1 is required for Oxr but not increased lifespan resulting from over-expression of DAF-16; concomitantly, DAF-16 over-expression rescues the short lifespan of skn-1 mutants but not their hypersensitivity to oxidative stress. These results suggest that SKN-1 promotes longevity by a mechanism other than protection against oxidative damage.

Andy's Algorithms: new automated digital image analysis pipelines for FIJI.

Quantification of cellular antigens and their interactions via antibody-based detection methods are widely used in scientific research. Accurate high-throughput quantitation of these assays using general image analysis software can be time consuming and challenging, particularly when attempted by users with limited image processing and analysis knowledge. To overcome this, we have designed Andy's Algorithms, a series of automated image analysis pipelines for FIJI, that permits rapid, accurate and reproducible batch-processing of 3,3'-diaminobenzidine (DAB) immunohistochemistry, proximity ligation assays (PLAs) and other common assays. Andy's Algorithms incorporates a step-by-step tutorial and optimization pipeline to make batch image analysis simple for the untrained user and adaptable across laboratories. Andy's algorithms provide a simpler, faster, standardized work flow compared to existing programs, while offering equivalent performance and additional features, in a free to use open-source application of FIJI. Andy's Algorithms are available at GitHub, publicly accessed at https://github.com/andlaw1841/Andy-s-Algorithm .