BRCA1 mutation carriers are predisposed to developing basal-like breast cancers with high metastasis and poor prognosis. Yet, how BRCA1 suppresses formation of basal-like breast cancers is still obscure. Deletion of p18 Ink4c (p18), an inhibitor of CDK4 and CDK6, functionally inactivates the RB pathway, stimulates mammary luminal stem cell (LSC) proliferation, and leads to spontaneous luminal tumor development. Alternately, germline mutation of Brca1 shifts the fate of luminal cells to cause luminal-to-basal mammary tumor transformation. Here, we report that disrupting Brca1 by either germline or epithelium-specific mutation in p18-deficient mice activates epithelial-to-mesenchymal transition (EMT) and induces dedifferentiation of LSCs, which associate closely with expansion of basal and cancer stem cells and formation of basal-like tumors. Mechanistically, BRCA1 bound to the TWIST promoter, suppressing its activity and inhibiting EMT in mammary tumor cells. In human luminal cancer cells, BRCA1 silencing was sufficient to activate TWIST and EMT and increase tumor formation. In parallel, TWIST expression and EMT features correlated inversely with BRCA1 expression in human breast cancers. Together, our findings showed that BRCA1 suppressed TWIST and EMT, inhibited LSC dedifferentiation, and repressed expansion of basal stem cells and basal-like tumors. Thus, our work offers the first genetic evidence that Brca1 directly suppresses EMT and LSC dedifferentiation during breast tumorigenesis. Cancer Res; 74(21); 6161-72. Ó2014 AACR.

It has been suggested that the small angle x-ray scattering (SAXS) pattern of human hair can be used to diagnose breast cancer and possibly to identify BRCA1/2 mutation carriers, who are at significantly elevated risk for developing breast cancer. In particular, the presence of a diffuse ring in the SAXS pattern was said to be diagnostic of either breast cancer or an increased risk thereof. To test this hypothesis, we measured SAXS from the pubic hair of 56 subjects with known BRCA1/2 and breast cancer status. We found that there is no clear association between the pattern of SAXS seen in human pubic hair and the risk of breast cancer or the presence of BRCA1/2 mutations. The possible use of SAXS to diagnose cancer remains conjectural, but this and previous studies do not suggest that SAXS can be used as a reliable method of identifying either BRCA1/2 mutation carriers or women who have had breast cancer.

Cryptococcal disease is estimated to affect nearly a quarter of a million people annually. Environmental isolates of Cryptococcus deneoformans, which make up 15 to 30% of clinical infections in temperate climates such as Europe, vary in their pathogenicity, ranging from benign to hyper-virulent. Key traits that contribute to virulence, such as the production of the pigment melanin, an extracellular polysaccharide capsule, and the ability to grow at human body temperature have been identified, yet little is known about the genetic basis of variation in such traits. Here we investigate the genetic basis of melanization, capsule size, thermal tolerance, oxidative stress resistance, and antifungal drug sensitivity using quantitative trait locus (QTL) mapping in progeny derived from a cross between two divergent C. deneoformans strains. Using a “function-valued” QTL analysis framework that exploits both time-series information and growth differences across multiple environments, we identified QTL for each of these virulence traits and drug susceptibility. For three QTL we identified the underlying genes and nucleotide differences that govern variation in virulence traits. One of these genes, RIC8, which encodes a regulator of cAMP-PKA signaling, contributes to variation in four virulence traits: melanization, capsule size, thermal tolerance, and resistance to oxidative stress. Two major effect QTL for amphotericin B resistance map to the genes SSK1 and SSK2, which encode key components of the HOG pathway, a fungal-specific signal transduction network that orchestrates cellular responses to osmotic and other stresses. We also discovered complex epistatic interactions within and between genes in the HOG and cAMP-PKA pathways that regulate antifungal drug resistance and resistance to oxidative stress. Our findings advance the understanding of virulence traits among diverse lineages of Cryptococcus, and highlight the role of genetic variation in key stress-responsive signaling pathways as a major contributor to phenotypic variation.

Sonic hedgehog signaling in the secondary heart field has a clear role in cardiac arterial pole development. In the absence of hedgehog signaling, proliferation is reduced in secondary heart field progenitors, and embryos predominantly develop pulmonary atresia. While it is expected that proliferation in the secondary heart field would be increased with elevated hedgehog signaling, this idea has never been tested. We hypothesized that up-regulating hedgehog signaling would increase secondary heart field proliferation, which would lead to arterial pole defects. In culture, secondary heart field explants proliferated up to 6-fold more in response to the hedgehog signaling agonist SAG, while myocardial differentiation and migration were unaffected. Treatment of chick embryos with SAG at HH14, just before the peak in secondary heart field proliferation, resulted unexpectedly in stenosis of both the aortic and pulmonary outlets. We examined proliferation in the secondary heart field and found that SAG-treated embryos exhibited a much milder increase in proliferation than was indicated by the in vitro experiments. To determine the source of other signaling factors that could modulate increased hedgehog signaling, we co-cultured secondary heart field explants with isolated pharyngeal endoderm or outflow tract and found that outflow tract co-cultures prevented SAG-induced proliferation. BMP2 is made and secreted by the outflow tract myocardium. To determine whether BMP signaling could prevent SAG-induced proliferation, we treated explants with SAG and BMP2 and found that BMP2 inhibited SAG-induced proliferation. In vivo, SAG-treated embryos showed up-regulated BMP2 expression and signaling. Together, these results indicate that BMP signaling from the outflow tract modulates hedgehog-induced proliferation in the secondary heart field.

BackgroundEstrogen promotes breast cancer development and progression mainly through estrogen receptor (ER). However, blockage of estrogen production or action prevents development of and suppresses progression of ER-negative breast cancers. How estrogen promotes ER-negative breast cancer development and progression is poorly understood. We previously discovered that deletion of cell cycle inhibitors p16Ink4a (p16) or p18Ink4c (p18) is required for development of Brca1-deficient basal-like mammary tumors, and that mice lacking p18 develop luminal-type mammary tumors.MethodsA genetic model system with three mouse strains, one that develops ER-positive mammary tumors (p18 single deletion) and the others that develop ER-negative tumors (p16;Brca1 and p18;Brca1 compound deletion), human BRCA1 mutant breast cancer patient-derived xenografts, and human BRCA1-deficient and BRCA1-proficient breast cancer cells were used to determine the role of estrogen in activating epithelial-mesenchymal transition (EMT), stimulating cell proliferation, and promoting ER-negative mammary tumor initiation and metastasis.ResultsEstrogen stimulated the proliferation and tumor-initiating potential of both ER-positive Brca1-proficient and ER-negative Brca1-deficient tumor cells. Estrogen activated EMT in a subset of Brca1-deficient mammary tumor cells that maintained epithelial features, and enhanced the number of cancer stem cells, promoting tumor progression and metastasis. Estrogen activated EMT independent of ER in Brca1-deficient, but not Brca1-proficient, tumor cells. Estrogen activated the AKT pathway in BRCA1-deficient tumor cells independent of ER, and pharmaceutical inhibition of AKT activity suppressed EMT and cell proliferation preventing BRCA1 deficient tumor progression.ConclusionsThis study reveals for the first time that estrogen promotes BRCA1-deficient tumor initiation and progression by stimulation of cell proliferation and activation of EMT, which are dependent on AKT activation and independent of ER.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-0996-9) contains supplementary material, which is available to authorized users.

Senescence prevents the proliferation of genomically damaged, but otherwise replication competent cells at risk of neoplastic transformation. p16INK4A (p16), an inhibitor of CDK4 and CDK6, plays a critical role in controlling cellular senescence in multiple organs. Functional inactivation of p16 by gene mutation and promoter methylation is frequently detected in human breast cancers. However, deleting p16 in mice or targeting DNA methylation within the murine p16 promoter does not result in mammary tumorigenesis. How loss of p16 contributes to mammary tumorigenesis in vivo is not fully understood.In this article, we reported that disruption of Brca1 in the mammary epithelium resulted in premature senescence that was rescued by p16 loss. We found that p16 loss transformed Brca1-deficient mammary epithelial cells and induced mammary tumors, though p16 loss alone was not sufficient to induce mammary tumorigenesis. We demonstrated that loss of both p16 and Brca1 led to metastatic, basal-like, mammary tumors with the induction of EMT and an enrichment of tumor initiating cells. We discovered that promoter methylation silenced p16 expression in most of the tumors developed in mice heterozygous for p16 and lacking Brca1. These data not only identified the function of p16 in suppressing BRCA1-deficient mammary tumorigenesis, but also revealed a collaborative effect of genetic mutation of p16 and epigenetic silencing of its transcription in promoting tumorigenesis. To the best of our knowledge, this is the first genetic evidence directly showing that p16 which is frequently deleted and inactivated in human breast cancers, collaborates with Brca1 controlling mammary tumorigenesis.

GATA3, a lineage specifier, controls lymphoid cell differentiation and its function in T cell commitment and development has been extensively studied. GATA3 promotes T cell specification by repressing B cell potential in pro T cells and decreased GATA3 expression is essential for early B cell commitment. Inherited genetic variation in GATA3 has been associated with lymphoma susceptibility. However, it remains elusive how the loss of function of GATA3 promotes B cell development and induces B cell lymphomas. In this study, we found that haploid loss of Gata3 by heterozygous germline deletion increased B cell populations in the bone marrow (BM) and spleen, and decreased CD4 T cell populations in the thymus, confirming that Gata3 promotes T and suppresses B cell development. We discovered that haploid loss of Gata3 reduced thymocyte proliferation with induction of p18Ink4c (p18), an inhibitor of CDK4 and CDK6, but enhanced B cell proliferation in the BM and spleen independent of p18. Loss of p18 partially restored Gata3 deficient thymocyte proliferation, but further stimulated Gata3 deficient B cell proliferation in the BM and spleen. Furthermore, we discovered that haploid loss of Gata3 in p18 deficient mice led to the development of B cell lymphomas that were capable of rapidly regenerating tumors when transplanted into immunocompromised mice. These results indicate that Gata3 deficiency promotes B cell differentiation and proliferation, and cooperates with p18 loss to induce B cell lymphomas. This study, for the first time, reveals that Gata3 is a tumor suppressor specifically in B cell lymphomagenesis.

Recent evidence indicates that the accumulation of endogenous DNA damage can induce senescence and limit the function of adult stem cells. It remains elusive whether deficiency in DNA damage repair is associated with the functional alteration of mammary stem cells. In this article, we reported that senescence was induced in mammary epithelial cells during aging along with increased expression of p16Ink4a (p16), an inhibitor of CDK4 and CKD6. Loss of p16 abrogated the age-induced senescence in mammary epithelial cells and significantly increased mammary stem cell function. We showed that loss of Brca1, a tumor suppressor that functions in DNA damage repair, in the mammary epithelium induced senescence with induction of p16 and a decline of stem cell function, which was rescued by p16 loss. These data not only answer the question as to whether deficiency in DNA damage repair is associated with the functional decline of mammary stem cells, but also identify the role of p16 in suppressing Brca1-deficient mammary stem cell function.