Genome-wide in vivo CRISPR screen identifies TGFß3 as actionable biomarker of palbociclib resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFß3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFß3 treatment can overcome this. This study defines TGFß3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFß3 to propose a new combinatorial treatment for TNBC.

Combined in vitro/in vivo genome-wide CRISPR screens in triple negative breast cancer identify cancer stemness regulators in paclitaxel resistance.

Triple negative breast cancer (TNBC) is defined as lacking the expressions of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC patients exhibit relatively poor clinical outcomes due to lack of molecular markers for targeted therapies. As such chemotherapy often remains the only systemic treatment option for these patients. While chemotherapy can initially help shrink TNBC tumor size, patients eventually develop resistance to drug, leading to tumor recurrence. We report a combined in vitro/in vivo genome-wide CRISPR synthetic lethality screening approach in a relevant TNBC cell line model to identify several targets responsible for the chemotherapy drug, paclitaxel resistance. Computational analysis integrating in vitro and in vivo data identified a set of genes, for which specific loss-of-function deletion enhanced paclitaxel resistance in TNBC. We found that several of these genes (ATP8B3, FOXR2, FRG2, HIST1H4A) act as cancer stemness negative regulators. Finally, using in vivo orthotopic transplantation TNBC models we showed that FRG2 gene deletion reduced paclitaxel efficacy and promoted tumor metastasis, while increasing FRG2 expression by means of CRISPR activation efficiently sensitized TNBC tumors to paclitaxel treatment and inhibited their metastatic abilities. In summary, the combined in vitro/in vivo genome-wide CRISPR screening approach proved effective as a tool to identify novel regulators of paclitaxel resistance/sensitivity and highlight the FRG2 gene as a potential therapeutical target overcoming paclitaxel resistance in TNBC.

In vivo genome-wide CRISPR screen reveals breast cancer vulnerabilities and synergistic mTOR/Hippo targeted combination therapy.

Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.

TGFß/cyclin D1/Smad-mediated inhibition of BMP4 promotes breast cancer stem cell self-renewal activity.

Basal-like triple-negative breast cancers (TNBCs) display poor prognosis, have a high risk of tumor recurrence, and exhibit high resistance to drug treatments. The TNBC aggressive features are largely due to the high proportion of cancer stem cells present within these tumors. In this study, we investigated the interplay and networking pathways occurring between TGFß family ligands in regulating stemness in TNBCs. We found that TGFß stimulation of TNBCs resulted in enhanced tumorsphere formation efficiency and an increased proportion of the highly tumorigenic CD44high/CD24low cancer stem cell population. Analysis of the TGFß transcriptome in TNBC cells revealed bone morphogenetic protein4 (BMP4) as a main TGFß-repressed target in these tumor cells. We further found that BMP4 opposed TGFß effects on stemness and potently decreased cancer stem cell numbers, thereby acting as a differentiation factor in TNBC. At the molecular level, we found that TGFß inhibition of BMP4 gene expression is mediated through the Smad pathway and cyclin D1. In addition, we also found BMP4 to act as a pro-differentiation factor in normal mammary epithelial cells and promote mammary acinar formation in 3D cell culture assays. Finally, and consistent with our in vitro results, in silico patient data analysis defined BMP4 as a potential valuable prognosis marker for TNBC patients.

Prolactin receptor-driven combined luminal and epithelial differentiation in breast cancer restricts plasticity, stemness, tumorigenesis and metastasis.

Dedifferentiation increased cellular plasticity and stemness are established derivers of tumor heterogeneity, metastasis and therapeutic failure resulting in incurable cancers. Therefore, it is essential to decipher pro/forward-differentiation mechanisms in cancer that may serve as therapeutic targets. We found that interfering with expression of the receptor for the lactogenic hormone prolactin (PRLR) in breast cancer cells representative of the luminal and epithelial breast cancer subtypes (hormone receptor positive (HR+) and HER2-enriched (HER2-E) resulted in loss of their differentiation state, enriched for stem-like cell subpopulations, and increased their tumorigenic capacity in a subtype-specific manner. Loss of PRLR expression in HR+ breast cancer cells caused their dedifferentiation generating a mesenchymal-basal-like phenotype enriched in CD44+ breast cancer stem-like cells (BCSCs) showing high tumorigenic and metastatic capacities and resistance to anti-hormonal therapy. Whereas loss of PRLR expression in HER2-E breast cancer cells resulted in loss of their luminal differentiation yet enriched for epithelial ALDH+ BCSC population showing elevated HER2-driven tumorigenic, multi-organ metastatic spread, and resistance to anti-HER2 therapy. Collectively, this study defines PRLR as a driver of precise luminal and epithelial differentiation limiting cellular plasticity, stemness, and tumorigenesis and emphasizing the function of pro/forward-differentiation pathways as a foundation for the discovery of anti-cancer therapeutic targets.

Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Dasatinib sensitises triple negative breast cancer cells to chemotherapy by targeting breast cancer stem cells.

BACKGROUND: Patients with triple negative breast cancer (TNBC) exhibit poor prognosis and are at high risk of tumour relapse, due to the resistance to chemotherapy. These aggressive phenotypes are in part attributed to the presence of breast cancer stem cells (BCSCs). Therefore, targeting BCSCs is a priority to overcoming chemotherapy failure in TNBCs. METHODS: We generated paclitaxel (pac)-resistant TNBC cells which displayed higher sphere forming potential and percentage of BCSC subpopulations compared to the parental cells. A screen with various kinase inhibitors revealed dasatinib, a Src kinase family inhibitor, as a potent suppressor of BCSC expansion/sphere formation in pac-resistant TNBC cells. RESULTS: We found dasatinib to block pac-induced BCSC enrichment and Src activation in both parental and pac-resistant TNBC cells. Interestingly, dasatinib induced an epithelial differentiation of the pac-resistant mesenchymal cells, resulting in their enhanced sensitivity to paclitaxel. The combination treatment of dasatinib and paclitaxel not only decreased the BCSCs numbers and their sphere forming capacity but also synergistically reduced cell viability of pac-resistant cells. Preclinical models of breast cancer further demonstrated the efficiency of the dasatinib/paclitaxel combination treatment in inhibiting tumour growth. CONCLUSIONS: Dasatinib is a promising anti-BCSC drug that could be used in combination with paclitaxel to overcome chemoresistance in TNBC.

Differential expression of TGFß isoforms in breast cancer highlights different roles during breast cancer progression.

While TGFß plays a critical role in tumor formation and progression, the role and contribution of its three different isoforms remain unclear. In this study, we aimed at elucidating the prognostic value of the TGFß isoforms and assessed their expression levels in breast cancer patients at different stages of the disease. We found higher levels of TGFß1 and TGFß3 in cancer patients compared to normal tissues, with no significant changes in TGFß2 expression. Similarly, TGFß1 and TGFß3, but not TGFß2, showed higher expression levels in advanced lymph node-positive and metastatic tumors, suggesting different roles for the different isoforms in tumor progression and the metastatic process, while in the least aggressive molecular subtype (luminal A), expression of the three TGFß isoforms significantly correlated with expression of both TGFß receptors, such correlation only occurred between TGFß1 and TGFß3 and the TGFß type II receptor (TßRII) in the highly aggressive basal-like subtype. Interestingly, a distinct and somehow opposite pattern was observed in HER-2 tumors, only showing significant association pattern between TGFß2 and the TGFß type I receptor (TßRI). Finally, the three TGFß isoforms showed distinct association patterns with patient outcome depending on the different molecular subtype, highlighting context-dependent, differential prognostic values.

KiSS1 gene as a novel mediator of TGFß-mediated cell invasion in triple negative breast cancer.

The invasive and metastatic phenotypes of breast cancer correlate with high recurrence rates and poor survival outcomes. Transforming growth factor-ß (TGFß) promotes tumor progression and metastasis in aggressive breast cancer. Here, we identified the kisspeptin KiSS1 as a downstream target of canonical TGFß/Smad2 pathway in triple negative breast cancer cells. We also found KiSS1 expression to be required for TGFß-induced cancer cell invasion. Indeed, knockdown expression of KiSS1 blocked TGFß-mediated cancer cell invasion as well as metalloproteinase (MMP9) expression and activity. Interestingly, Kisspeptin-10 (KP-10), the smallest active form of kisspeptin also stimulates cancer cell invasive behavior through activation of MAPK/Erk pathway. We described a positive feedback loop between KiSS1 and p21 downstream of TGFß, further contributing to TGFß-induced cancer cell invasion. Lastly, we explored both the clinical utility of KiSS1 as a lymph node involvement predictive tool and its potential as a therapeutic target. We found KiSS1 high expression to correlate with lymph node positive status. Furthermore, blocking KiSS1 using a specific small peptide antagonist (p234) impaired TGFß-mediated cell invasion and MMP9 induction. Together, our results define an essential role of KiSS1 in regulating TGFß pro-invasive effects and define KiSS1 as a therapeutic new target for triple negative breast cancer.

Cyclooxygenase-2 regulates TGFß-induced cancer stemness in triple-negative breast cancer.

Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, display poor prognosis and exhibit resistance to conventional therapies, partly due to an enrichment in breast cancer stem cells (BCSCs). Here, we investigated the role of the cyclooxygenase-2 (COX-2), a downstream target of TGFß, in regulating BCSCs in TNBC. Bioinformatics analysis revealed that COX-2 is highly expressed in TNBC and that its expression correlated with poor survival outcome in basal subtype of breast cancer. We also found TGFß-mediated COX-2 expression to be Smad3-dependent and to be required for BCSC self-renewal and expansion in TNBCs. Knocking down COX-2 expression strikingly blocked TGFß-induced tumorsphere formation and TGFß-induced enrichment of the two stem-like cell populations, CD24lowCD44high and ALDH+ BCSCs. Blocking COX-2 activity, using a pharmacological inhibitor also prevented TGFß-induced BCSC self-renewal. Moreover, we found COX-2 to be required for TGFß-induced expression of mesenchymal and basal breast cancer markers. In particular, we found that TGFß-induced expression of fibronectin plays a central role in TGFß-mediated breast cancer stemness. Together, our results describe a novel role for COX-2 in mediating the TGFß effects on BCSC properties and imply that targeting the COX-2 pathway may prove useful for the treatment of TNBC by eliminating BCSCs.

CDK4 regulates cancer stemness and is a novel therapeutic target for triple-negative breast cancer.

Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors.

Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor ß/Smad signaling and associates with favorable breast cancer disease outcomes.

INTRODUCTION: This study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor ß (TGFß) pathway, which induces potent migration and invasion responses in breast cancer cells. METHODS: We surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFß/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFß-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging-based matrix degradation assay, respectively. RESULTS: Relatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFß through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFß/Smad signaling pathway. CONCLUSION: BCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFß/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3's molecular mechanism of action and highlight BCAR3 as a novel TGFß/Smad antagonist in breast cancer.

Cyclin D1 cooperates with p21 to regulate TGFß-mediated breast cancer cell migration and tumor local invasion.

INTRODUCTION: Deregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFß) in breast cancer progression. METHODS: Cyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion. RESULTS: We found TGFß to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFß-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFß-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFß promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21. CONCLUSIONS: Thus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFß-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.

MicroRNA-584 and the protein phosphatase and actin regulator 1 (PHACTR1), a new signaling route through which transforming growth factor-ß Mediates the migration and actin dynamics of breast cancer cells.

TGF-ß plays an important role in breast cancer progression as a prometastatic factor, notably through enhancement of cell migration. It is becoming clear that microRNAs, a new class of small regulatory molecules, also play crucial roles in mediating tumor formation and progression. We found TGF-ß to down-regulate the expression of the microRNA miR-584 in breast cancer cells. Furthermore, we identified PHACTR1, an actin-binding protein, to be positively regulated by TGF-ß in a miR-584-dependent manner. Moreover, we found TGF-ß-mediated down-regulation of miR-584 and increased expression of PHACTR1 to be required for TGF-ß-induced cell migration of breast cancer cells. Indeed, both overexpression of miR-584 and knockdown of PHACTR1 resulted in a drastic reorganization of the actin cytoskeleton and reduced TGF-ß-induced cell migration. Our data highlight a novel signaling route whereby TGF-ß silences the expression of miR-584, resulting in enhanced PHACTR1 expression, and further leading to actin rearrangement and breast cancer cell migration.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFß-mediated breast cancer cell migration and invasion.

INTRODUCTION: Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFß)-mediated breast cancer cell migration and invasion. METHODS: A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFß and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFß transcriptional activity was measured by a TGFß/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFß downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFß/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive. RESULTS: We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFß. p21 and p/CAF regulates TGFß-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFß/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients. CONCLUSIONS: Together these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

Engulfment protein GULP is regulator of transforming growth factor-ß response in ovarian cells.

Transforming growth factor ß (TGF-ß) is a key regulatory molecule with pleiotropic effects on cell growth, migration, and invasion. As a result, impairment of proper TGF-ß signaling is central to tumorigenesis and metastasis. The TGF-ß receptor V (TGFBRV or LRP1) has been shown to be responsible for TGF-ß-mediated cell growth inhibition in Chinese hamster ovary (CHO) cells. The LRP1 adapter protein GULP mediates internalization of the various LRP1-specific ligands, and we hypothesize that GULP acts as a novel regulator of TGF-ß signaling in ovarian cells. CHO cells that overexpress exogenous GULP (FL) demonstrate enhancement in growth inhibition, migration, and invasion from TGF-ß treatment, whereas cells that lack GULP (AS) show impairment of growth inhibition and decreased migration and invasion. The enhanced TGF-ß response in FL cells was confirmed by a prolonged TGF-ß-induced SMAD3 phosphorylation, whereas a shortening of the phosphorylation event is observed in AS cells. Mechanistically, the presence of GULP retains the TGF-ß in a signaling-competent early endosome for enhanced signaling. To address this mechanism in a physiological setting, TGF-ß insensitive ovarian adenocarcinoma cells (HEY) have a very low GULP expression level, similar to the observation made in a wide selection of human ovarian adenocarcinomas. Transfection of GULP into the HEY cells restored the TGF-ß responsiveness, as measured by SMAD3 phosphorylation and impairment of cell growth. Because GULP expression positively regulates TGF-ß signaling leading to growth inhibition, this may represent an attractive target to achieve TGF-ß responsiveness in ovarian cells.

Smad signaling antagonizes STAT5-mediated gene transcription and mammary epithelial cell differentiation.

Both the transforming growth factor-beta (TGFbeta)/Smad and the prolactin/JAK/STAT pathway are critical to the proper development, maintenance, and function of the mammary epithelial tissue. Interestingly, opposing physiological effects between these two signaling pathways are prominent in the regulation of mammary gland development. However, the exact nature of the biological network existing between the Smad and STAT signal transduction pathways has remained elusive. We identified a novel regulatory cross-talk mechanism by which TGFbeta-induced Smad signaling acts to antagonize prolactin-mediated JAK/STAT signaling and expression of target genes. Furthermore, we found activin, another member of the TGFbeta family, to also efficiently block STAT5 signaling and beta-casein expression in mammary epithelial cells. Our results indicate that ligand-induced activation of Smad2, -3, and -4 by activin and TGFbeta leads to a direct inhibition of STAT5 transactivation and STAT5-mediated transcription of the downstream target genes, beta-casein and cyclin D1, thereby blocking vital processes for mammary gland growth and differentiation. Finally, we unveiled the mechanism by which these two signaling cascades antagonize their effects, and we found that activated Smads inhibit STAT5 association with its co-activator CREB-binding protein, thus blocking STAT5 transactivation of its target genes and leading to inhibition of mammary gland differentiation and lactation.