GATA3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in breast cancer.

BACKGROUND: Inadequate DNA damage repair promotes aberrant differentiation of mammary epithelial cells. Mammary luminal cell fate is mainly determined by a few transcription factors including GATA3. We previously reported that GATA3 functions downstream of BRCA1 to suppress aberrant differentiation in breast cancer. How GATA3 impacts DNA damage repair preventing aberrant cell differentiation in breast cancer remains elusive. We previously demonstrated that loss of p18, a cell cycle inhibitor, in mice induces luminal-type mammary tumors, whereas depletion of either Brca1 or Gata3 in p18 null mice leads to basal-like breast cancers (BLBCs) with activation of epithelial-mesenchymal transition (EMT). We took advantage of these mutant mice to examine the role of Gata3 as well as the interaction of Gata3 and Brca1 in DNA damage repair in mammary tumorigenesis. RESULTS: Depletion of Gata3, like that of Brca1, promoted DNA damage accumulation in breast cancer cells in vitro and in basal-like breast cancers in vivo. Reconstitution of Gata3 improved DNA damage repair in Brca1-deficient mammary tumorigenesis. Overexpression of GATA3 promoted homologous recombination (HR)-mediated DNA damage repair and restored HR efficiency of BRCA1-deficient cells. Depletion of Gata3 sensitized tumor cells to PARP inhibitor (PARPi), and reconstitution of Gata3 enhanced resistance of Brca1-deficient tumor cells to PARP inhibitor. CONCLUSIONS: These results demonstrate that Gata3 functions downstream of BRCA1 to promote DNA damage repair and suppress dedifferentiation in mammary tumorigenesis and progression. Our findings suggest that PARP inhibitors are effective for the treatment of GATA3-deficient BLBCs.

PAPAS promotes differentiation of mammary epithelial cells and suppresses breast carcinogenesis.

Extensive remodeling of the female mammary epithelium during development and pregnancy has been linked to cancer susceptibility. The faithful response of mammary epithelial cells (MECs) to hormone signaling is key to avoiding breast cancer development. Here, we show that lactogenic differentiation of murine MECs requires silencing of genes encoding ribosomal RNA (rRNA) by the antisense transcript PAPAS. Accordingly, knockdown of PAPAS derepresses rRNA genes, attenuates the response to lactogenic hormones, and induces malignant transformation. Restoring PAPAS levels in breast cancer cells reduces tumorigenicity and lung invasion and activates many interferon-regulated genes previously linked to metastasis suppression. Mechanistically, PAPAS transcription depends on R-loop formation at the 3' end of rRNA genes, which is repressed by RNase H1 and replication protein A (RPA) overexpression in breast cancer cells. Depletion of PAPAS and upregulation of RNase H1 and RPA in human breast cancer underpin the clinical relevance of our findings.

The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair.

Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.

p18INK4C and BRCA1 inhibit follicular cell proliferation and dedifferentiation in thyroid cancer.

Only 3% of thyroid cancers are medullary thyroid carcinomas (MTCs), the rest are follicular epithelial cell derived non-MTCs (NMTCs). A dysfunctional INK4-CDK4-RB pathway is detected in most of NMTCs. DNA repair defects and genome instability are associated with NMTC dedifferentiation and aggressiveness. Whether inactivation of the INK4-CDK4-RB pathway induces NMTCs and how differentiation of NMTC cells is controlled remain elusive. In this study, we generated p18Ink4c and Brca1 singly and doubly deficient mice as well as p16Ink4a and Brca1 singly and doubly deficient mice. By using these mice and human thyroid carcinoma cell lines, we discovered that loss of p18Ink4c, not p16Ink4a, in mice stimulated follicular cell proliferation and induced NMTCs. Depletion of Brca1 alone or both p16Ink4a and Brca1 did not induce thyroid tumor. Depletion of Brca1 in p18Ink4c null mice results in poorly differentiated and aggressive NMTCs with epithelial-mesenchymal transition (EMT) features and enhanced DNA damage. Knockdown of BRCA1 in thyroid carcinoma cells activated EMT and promoted tumorigenesis whereas overexpression of BRCA1 inhibited EMT. BRCA1 and EMT marker expression were inversely related in human thyroid cancers. Our finding, for the first time, demonstrates that inactivation of INK4-CDK4-RB pathway induces NMTCs and that Brca1 deficiency promotes dedifferentiation of NMTC cells. These results suggest that BRCA1 and p18INK4C collaboratively suppress thyroid tumorigenesis and progression and CDK4 inhibitors will be effective for treatment of INK4-inactivated or cyclin D-overexpressed thyroid carcinomas.

Loss of function of GATA3 regulates FRA1 and c-FOS to activate EMT and promote mammary tumorigenesis and metastasis.

Basal-like breast cancers (BLBCs) are among the most aggressive cancers, partly due to their enrichment of cancer stem cells (CSCs). Breast CSCs can be generated from luminal-type cancer cells via epithelial-mesenchymal transition (EMT). GATA3 maintains luminal cell fate, and its expression is lost or reduced in BLBCs. However, deletion of Gata3 in mice or cells results in early lethality or proliferative defects. It is unknown how loss-of-function of GATA3 regulates EMT and CSCs in breast cancer. We report here that haploid loss of Gata3 in mice lacking p18Ink4c, a cell cycle inhibitor, up-regulates Fra1, an AP-1 family protein that promotes mesenchymal traits, and downregulates c-Fos, another AP-1 family protein that maintains epithelial fate, leading to activation of EMT and promotion of mammary tumor initiation and metastasis. Depletion of Gata3 in luminal tumor cells similarly regulates Fra1 and c-Fos in activation of EMT. GATA3 binds to FOSL1 (encoding FRA1) and FOS (encoding c-FOS) loci to repress FOSL1 and activate FOS transcription. Deletion of Fra1 or reconstitution of Gata3, but not reconstitution of c-Fos, in Gata3 deficient tumor cells inhibits EMT, preventing tumorigenesis and/or metastasis. In human breast cancers, GATA3 expression is negatively correlated with FRA1 and positively correlated with c-FOS. Low GATA3 and FOS, but high FOSL1, are characteristics of BLBCs. Together, these data provide the first genetic evidence indicating that loss of function of GATA3 in mammary tumor cells activates FOSL1 to promote mesenchymal traits and CSC function, while concurrently repressing FOS to lose epithelial features. We demonstrate that FRA1 is required for the activation of EMT in GATA3 deficient tumorigenesis and metastasis.

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer.

5'-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.

Loss of function of BRCA1 promotes EMT in mammary tumors through activation of TGFßR2 signaling pathway.

BRCA1 deficient breast cancers are aggressive and chemoresistant due, in part, to their enrichment of cancer stem cells that can be generated from carcinoma cells by an epithelial-mesenchymal transition (EMT). We previously discovered that BRCA1 deficiency activates EMT in mammary tumorigenesis. How BRCA1 controls EMT and how to effectively target BRCA1-deficient cancers remain elusive. We analyzed murine and human tumors and identified a role for Tgfßr2 in governing the molecular aspects of EMT that occur with Brca1 loss. We utilized CRISPR to delete Tgfßr2 and specific inhibitors to block Tgfßr2 activity and followed up with the molecular analysis of assays for tumor growth and metastasis. We discovered that heterozygous germline deletion, or epithelia-specific deletion of Brca1 in mice, activates Tgfßr2 signaling pathways in mammary tumors. BRCA1 depletion promotes TGFß-mediated EMT activation in cancer cells. BRCA1 binds to the TGFßR2 locus to repress its transcription. Targeted deletion or pharmaceutical inhibition of Tgfßr2 in Brca1-deficient tumor cells reduces EMT and suppresses tumorigenesis and metastasis. BRCA1 and TGFßR2 expression levels are inversely related in human breast cancers. This study reveals for the first time that a targetable TGFßR signaling pathway is directly activated by BRCA1-deficiency in the induction of EMT in breast cancer progression.

Loss of function of GATA3 induces basal-like mammary tumors.

Purpose: GATA3 is a transcription factor essential for mammary luminal epithelial cell differentiation. Expression of GATA3 is absent or significantly reduced in basal-like breast cancers. Gata3 loss-of-function impairs cell proliferation, making it difficult to investigate the role of GATA3 deficiency in vivo. We previously demonstrated that CDK inhibitor p18INK4c (p18) is a downstream target of GATA3 and restrains mammary epithelial cell proliferation and tumorigenesis. Whether and how loss-of-function of GATA3 results in basal-like breast cancers remains elusive. Methods: We generated mutant mouse strains with heterozygous germline deletion of Gata3 in p18 deficient backgrounds and developed a Gata3 depleted mammary tumor model system to determine the role of Gata3 loss in controlling cell proliferation and aberrant differentiation in mammary tumor development and progression. Results: Haploid loss of Gata3 reduced mammary epithelial cell proliferation with induction of p18, impaired luminal differentiation, and promoted basal differentiation in mammary glands. p18 deficiency induced luminal type mammary tumors and rescued the proliferative defect caused by haploid loss of Gata3. Haploid loss of Gata3 accelerated p18 deficient mammary tumor development and changed the properties of these tumors, resulting in their malignant and luminal-to-basal transformation. Expression of Gata3 negatively correlated with basal differentiation markers in MMTV-PyMT mammary tumor cells. Depletion of Gata3 in luminal tumor cells also reduced cell proliferation with induction of p18 and promoted basal differentiation. We confirmed that expression of GATA3 and basal markers are inversely correlated in human basal-like breast cancers. Conclusions: This study provides the first genetic evidence demonstrating that loss-of-function of GATA3 directly induces basal-like breast cancer. Our finding suggests that basal-like breast cancer may also originate from luminal type cancer.

GATA3 functions downstream of BRCA1 to suppress EMT in breast cancer.

Purpose: Functional loss of BRCA1 is associated with poorly differentiated and metastatic breast cancers that are enriched with cancer stem cells (CSCs). CSCs can be generated from carcinoma cells through an epithelial-mesenchymal transition (EMT) program. We and others have previously demonstrated that BRCA1 suppresses EMT and regulates the expression of multiple EMT-related transcription factors. However, the downstream mediators of BRCA1 function in EMT suppression remain elusive. Methods: Depletion of BRCA1 or GATA3 activates p18INK4C , a cell cycle inhibitor which inhibits mammary epithelial cell proliferation. We have therefore created genetically engineered mice with Brca1 or Gata3 loss in addition to deletion of p18INK4C , to rescue proliferative defects caused by deficiency of Brca1 or Gata3. By using these mutant mice along with human BRCA1 deficient as well as proficient breast cancer tissues and cells, we investigated and compared the role of Brca1 and Gata3 loss in the activation of EMT in breast cancers. Results: We discovered that BRCA1 and GATA3 expressions were positively correlated in human breast cancer. Depletion of BRCA1 stimulated methylation of GATA3 promoter thereby repressing GATA3 transcription. We developed Brca1 and Gata3 deficient mouse system. We found that Gata3 deficiency in mice induced poorly-differentiated mammary tumors with the activation of EMT and promoted tumor initiating and metastatic potential. Gata3 deficient mammary tumors phenocopied Brca1 deficient tumors in the induction of EMT under the same genetic background. Reconstitution of Gata3 in Brca1-deficient tumor cells activated mesenchymal-epithelial transition, suppressing tumor initiation and metastasis. Conclusions: Our finding, for the first time, demonstrates that GATA3 functions downstream of BRCA1 to suppress EMT in controlling mammary tumorigenesis and metastasis.

PDGFRß is an essential therapeutic target for BRCA1-deficient mammary tumors.

BACKGROUND: Basal-like breast cancers (BLBCs) are a leading cause of cancer death due to their capacity to metastasize and lack of effective therapies. More than half of BLBCs have a dysfunctional BRCA1. Although most BRCA1-deficient cancers respond to DNA-damaging agents, resistance and tumor recurrence remain a challenge to survival outcomes for BLBC patients. Additional therapies targeting the pathways aberrantly activated by BRCA1 deficiency are urgently needed. METHODS: Most BRCA1-deficient BLBCs carry a dysfunctional INK4-RB pathway. Thus, we created genetically engineered mice with Brca1 loss and deletion of p16INK4A, or separately p18INK4C, to model the deficient INK4-RB signaling in human BLBC. By using these mutant mice and human BRCA1-deficient and proficient breast cancer tissues and cells, we tested if there exists a druggable target in BRCA1-deficient breast cancers. RESULTS: Heterozygous germline or epithelium-specific deletion of Brca1 in p18INK4C- or p16INK4A-deficient mice activated Pdgfrß signaling, induced epithelial-to-mesenchymal transition, and led to BLBCs. Confirming this role, targeted deletion of Pdgfrß in Brca1-deficient tumor cells promoted cell death, induced mesenchymal-to-epithelial transition, and suppressed tumorigenesis. Importantly, we also found that pharmaceutical inhibition of Pdgfrß and its downstream target Pkcα suppressed Brca1-deficient tumor initiation and progression and effectively killed BRCA1-deficient cancer cells. CONCLUSIONS: Our work offers the first genetic and biochemical evidence that PDGFRß-PKCα signaling is repressed by BRCA1, which establishes PDGFRß-PKCα signaling as a therapeutic target for BRCA1-deficient breast cancers.

FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange.

FANCA is a component of the Fanconi anemia (FA) core complex that activates DNA interstrand crosslink repair by monoubiquitination of FANCD2. Here, we report that purified FANCA protein catalyzes bidirectional single-strand annealing (SA) and strand exchange (SE) at a level comparable to RAD52, while a disease-causing FANCA mutant, F1263Δ, is defective in both activities. FANCG, which directly interacts with FANCA, dramatically stimulates its SA and SE activities. Alternatively, FANCB, which does not directly interact with FANCA, does not stimulate this activity. Importantly, five other patient-derived FANCA mutants also exhibit deficient SA and SE, suggesting that the biochemical activities of FANCA are relevant to the etiology of FA. A cell-based DNA double-strand break (DSB) repair assay demonstrates that FANCA plays a direct role in the single-strand annealing sub-pathway (SSA) of DSB repair by catalyzing SA, and this role is independent of the canonical FA pathway and RAD52.

Estrogen promotes estrogen receptor negative BRCA1-deficient tumor initiation and progression.

BACKGROUND: Estrogen 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. METHODS: A 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. RESULTS: Estrogen 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. CONCLUSIONS: This 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.

p16 loss rescues functional decline of Brca1-deficient mammary stem cells.

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.

p16INK4a suppresses BRCA1-deficient mammary tumorigenesis.

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 restrains B cell proliferation and cooperates with p18INK4c to repress B cell lymphomagenesis.

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.

The BET bromodomain inhibitor I-BET151 acts downstream of smoothened protein to abrogate the growth of hedgehog protein-driven cancers.

Epigenetic enzymes modulate signal transduction pathways in different biological contexts. We reasoned that epigenetic regulators might modulate the Hedgehog (HH) signaling pathway, a main driver of cell proliferation in various cancers including medulloblastoma. To test this hypothesis, we performed an unbiased small-molecule screen utilizing an HH-dependent reporter cell line (Light2 cells). We incubated Light2 cells with small molecules targeting different epigenetic modulators and identified four histone deacetylase inhibitors and a bromodomain and extra terminal domain (BET) protein inhibitor (I-BET151) that attenuate HH activity. I-BET151 was also able to inhibit the expression of HH target genes in Sufu(-/-) mouse embryonic fibroblasts, in which constitutive Gli activity is activated in a Smoothened (Smo)-independent fashion, consistent with it acting downstream of Smo. Knockdown of Brd4 (which encodes one of the BET proteins) phenocopies I-BET151 treatment, suggesting that Brd4 is a regulator of the HH signaling pathway. Consistent with this suggestion, Brd4 associates with the proximal promoter region of the Gli1 locus, and does so in a manner that can be reversed by I-BET151. Importantly, I-BET151 also suppressed the HH activity-dependent growth of medulloblastoma cells, in vitro and in vivo. These studies suggest that BET protein modulation may be an attractive therapeutic strategy for attenuating the growth of HH-dependent cancers, such as medulloblastoma.

BRCA1 suppresses epithelial-to-mesenchymal transition and stem cell dedifferentiation during mammary and tumor development.

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.

Notch signaling drives stemness and tumorigenicity of esophageal adenocarcinoma.

Esophageal adenocarcinoma ranks sixth in cancer mortality in the world and its incidence has risen dramatically in the Western population over the last decades. Data presented herein strongly suggest that Notch signaling is critical for esophageal adenocarcinoma and underlies resistance to chemotherapy. We present evidence that Notch signaling drives a cancer stem cell phenotype by regulating genes that establish stemness. Using patient-derived xenograft models, we demonstrate that inhibition of Notch by gamma-secretase inhibitors (GSI) is efficacious in downsizing tumor growth. Moreover, we demonstrate that Notch activity in a patient's ultrasound-assisted endoscopic-derived biopsy might predict outcome to chemotherapy. Therefore, this study provides a proof of concept that inhibition of Notch activity will have efficacy in treating esophageal adenocarcinoma, offering a rationale to lay the foundation for a clinical trial to evaluate the efficacy of GSI in esophageal adenocarcinoma treatment.

Pyrvinium attenuates Hedgehog signaling downstream of smoothened.

The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds, vismodegib, is now FDA-approved for patients with advanced basal cell carcinoma, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50, 10 nmol/L) casein kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib -resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator suppressor of fused. We go on to demonstrate the utility of this small molecule in vivo, against the HH-dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers.

The E3 ligase PARC mediates the degradation of cytosolic cytochrome c to promote survival in neurons and cancer cells.

The ability to withstand mitochondrial damage is especially critical for the survival of postmitotic cells, such as neurons. Likewise, cancer cells can also survive mitochondrial stress. We found that cytochrome c (Cyt c), which induces apoptosis upon its release from damaged mitochondria, is targeted for proteasome-mediated degradation in mouse neurons, cardiomyocytes, and myotubes and in human glioma and neuroblastoma cells, but not in proliferating human fibroblasts. In mouse neurons, apoptotic protease-activating factor 1 (Apaf-1) prevented the proteasome-dependent degradation of Cyt c in response to induced mitochondrial stress. An RNA interference screen in U-87 MG glioma cells identified p53-associated Parkin-like cytoplasmic protein (PARC, also known as CUL9) as an E3 ligase that targets Cyt c for degradation. The abundance of PARC positively correlated with differentiation in mouse neurons, and overexpression of PARC reduced the abundance of mitochondrially-released cytosolic Cyt c in various cancer cell lines and in mouse embryonic fibroblasts. Conversely, neurons from Parc-deficient mice had increased sensitivity to mitochondrial damage, and neuroblastoma or glioma cells in which PARC or ubiquitin was knocked down had increased abundance of mitochondrially-released cytosolic Cyt c and decreased viability in response to stress. These findings suggest that PARC-mediated ubiquitination and degradation of Cyt c is a strategy engaged by both neurons and cancer cells to prevent apoptosis during conditions of mitochondrial stress.