p27 transcriptionally coregulates cJun to drive programs of tumor progression.

p27 shifts from CDK inhibitor to oncogene when phosphorylated by PI3K effector kinases. Here, we show that p27 is a cJun coregulator, whose assembly and chromatin association is governed by p27 phosphorylation. In breast and bladder cancer cells with high p27pT157pT198 or expressing a CDK-binding defective p27pT157pT198 phosphomimetic (p27CK-DD), cJun is activated and interacts with p27, and p27/cJun complexes localize to the nucleus. p27/cJun up-regulates TGFB2 to drive metastasis in vivo. Global analysis of p27 and cJun chromatin binding and gene expression shows that cJun recruitment to many target genes is p27 dependent, increased by p27 phosphorylation, and activates programs of epithelial-mesenchymal transformation and metastasis. Finally, human breast cancers with high p27pT157 differentially express p27/cJun-regulated genes of prognostic relevance, supporting the biological significance of the work.

C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer.

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

Protein kinase Cδ negatively regulates Notch1-dependent transcription via a kinase-independent mechanism in vitro.

Protein kinase Cδ (PKCδ) plays a significant role in the regulation of growth, apoptosis, and differentiation in a diversity of cell types. We investigated the effect of PKCδ on Notch1 intracellular domain (NICD)-mediated transcription with Notch transcription reporter constructs. The results indicate that co-expression of PKCδ down-regulated NICD-dependent transcription. Co-expression of a dominant negative PKCδ (K376R) variant lacking kinase activity was also able to downregulate NICD-dependent transcription, suggesting that PKCδ exerts its inhibitory effect via a kinase-independent mechanism(s). Interestingly, expression of PKCδ as well as K376R induced NICD up-regulation by inhibiting proteasome-mediated degradation of NICD, indicating that NICD protein quantity is not proportional to its transcriptional activity. When the subcellular distribution of NICD was investigated by both subcellular fractionation and immunocytochemistry, it was found that PKCδ and K376R effectively impaired proper nuclear localization of NICD, possibly via a physical association between NICD and PKCδ, which was confirmed by co-immunoprecipitation experiments. Chromatin immunoprecipitation assays revealed that both PKCδ and K376R inhibit the association of NICD with the promoter region of its target gene, Hes1. Furthermore, silencing of PKCδ resulted in increased NICD nuclear localization and NICD transcriptional activity in MCF-7 cells. PKCδ silencing-induced increase in anti-apoptotic survivin could not rescue apoptosis induced by doxorubicin. The data herein indicate that PKCδ can induce down-regulation of NICD transcriptional activity via a kinase-independent inhibition of NICD nuclear targeting and dissociation of NICD from target gene promoters.

CD24 enhances DNA damage-induced apoptosis by modulating NF-κB signaling in CD44-expressing breast cancer cells.

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-κB) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-κB signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-κB signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-κB signaling in CD44-expressing cells.

ATP-Competitive Inhibitors Midostaurin and Avapritinib Have Distinct Resistance Profiles in Exon 17-Mutant KIT.

KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17-mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations. SIGNIFICANCE: This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.

Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells.

Purpose: Rational targeted therapies are needed for treatment of ovarian cancers. Signaling kinases Src and MAPK are activated in high-grade serous ovarian cancer (HGSOC). Here, we tested the frequency of activation of both kinases in HGSOC and the therapeutic potential of dual kinase inhibition.Experimental Design: MEK and Src activation was assayed in primary HGSOC from The Cancer Genome Atlas (TGGA). Effects of dual kinase inhibition were assayed on cell-cycle, apoptosis, gene, and proteomic analysis; cancer stem cells; and xenografts.Results: Both Src and MAPK are coactivated in 31% of HGSOC, and this associates with worse overall survival on multivariate analysis. Frequent dual kinase activation in HGSOC led us to assay the efficacy of combined Src and MEK inhibition. Treatment of established lines and primary ovarian cancer cultures with Src and MEK inhibitors saracatinib and selumetinib, respectively, showed target kinase inhibition and synergistic induction of apoptosis and cell-cycle arrest in vitro, and tumor inhibition in xenografts. Gene expression and proteomic analysis confirmed cell-cycle inhibition and autophagy. Dual therapy also potently inhibited tumor-initiating cells. Src and MAPK were both activated in tumor-initiating populations. Combination treatment followed by drug washout decreased sphere formation and ALDH1+ cells. In vivo, tumors dissociated after dual therapy showed a marked decrease in ALDH1 staining, sphere formation, and loss of tumor-initiating cells upon serial xenografting.Conclusions: Selumetinib added to saracatinib overcomes EGFR/HER2/ERBB2-mediated bypass activation of MEK/MAPK observed with saracatinib alone and targets tumor-initiating ovarian cancer populations, supporting further evaluation of combined Src-MEK inhibition in clinical trials. Clin Cancer Res; 24(19); 4874-86. ©2018 AACR.

VEGFA activates an epigenetic pathway upregulating ovarian cancer-initiating cells.

The angiogenic factor, VEGFA, is a therapeutic target in ovarian cancer (OVCA). VEGFA can also stimulate stem-like cells in certain cancers, but mechanisms thereof are poorly understood. Here, we show that VEGFA mediates stem cell actions in primary human OVCA culture and OVCA lines via VEGFR2-dependent Src activation to upregulate Bmi1, tumor spheres, and ALDH1 activity. The VEGFA-mediated increase in spheres was abrogated by Src inhibition or SRC knockdown. VEGFA stimulated sphere formation only in the ALDH1+ subpopulation and increased OVCA-initiating cells and tumor formation in vivo through Bmi1. In contrast to its action in hemopoietic malignancies, DNA methyl transferase 3A (DNMT3A) appears to play a pro-oncogenic role in ovarian cancer. VEGFA-driven Src increased DNMT3A leading to miR-128-2 methylation and upregulation of Bmi1 to increase stem-like cells. SRC knockdown was rescued by antagomir to miR-128. DNMT3A knockdown prevented VEGFA-driven miR-128-2 loss, and the increase in Bmi1 and tumor spheres. Analysis of over 1,300 primary human OVCAs revealed an aggressive subset in which high VEGFA is associated with miR-128-2 loss. Thus, VEGFA stimulates OVCA stem-like cells through Src-DNMT3A-driven miR-128-2 methylation and Bmi1 upregulation.

Interactions between Adipocytes and Breast Cancer Cells Stimulate Cytokine Production and Drive Src/Sox2/miR-302b-Mediated Malignant Progression.

Consequences of the obesity epidemic on cancer morbidity and mortality are not fully appreciated. Obesity is a risk factor for many cancers, but the mechanisms by which it contributes to cancer development and patient outcome have yet to be fully elucidated. Here, we examined the effects of coculturing human-derived adipocytes with established and primary breast cancer cells on tumorigenic potential. We found that the interaction between adipocytes and cancer cells increased the secretion of proinflammatory cytokines. Prolonged culture of cancer cells with adipocytes or cytokines increased the proportion of mammosphere-forming cells and of cells expressing stem-like markers in vitro. Furthermore, contact with immature adipocytes increased the abundance of cancer cells with tumor-forming and metastatic potential in vivo. Mechanistic investigations demonstrated that cancer cells cultured with immature adipocytes or cytokines activated Src, thus promoting Sox2, c-Myc, and Nanog upregulation. Moreover, Sox2-dependent induction of miR-302b further stimulated cMYC and SOX2 expression and potentiated the cytokine-induced cancer stem cell-like properties. Finally, we found that Src inhibitors decreased cytokine production after coculture, indicating that Src is not only activated by adipocyte or cytokine exposures, but is also required to sustain cytokine induction. These data support a model in which cancer cell invasion into local fat would establish feed-forward loops to activate Src, maintain proinflammatory cytokine production, and increase tumor-initiating cell abundance and metastatic progression. Collectively, our findings reveal new insights underlying increased breast cancer mortality in obese individuals and provide a novel preclinical rationale to test the efficacy of Src inhibitors for breast cancer treatment.

CD24 regulates cell proliferation and transforming growth factor ß-induced epithelial to mesenchymal transition through modulation of integrin ß1 stability.

To determine the role of CD24 in breast cancer cells, we knocked down CD24 in MCF-7 human breast cancer cells by retroviral delivery of shRNA. MCF-7 cells with knocked down CD24 (MCF-7 hCD24 shRNA) exhibited decreased cell proliferation and cell adhesion as compared to control MCF-7 mCD24 shRNA cells. Decreased proliferation of MCF-7 hCD24 shRNA cells resulted from the inhibition of cell cycle progression from G1 to S phase. The specific inhibition of MEK/ERK signaling by CD24 ablation might be responsible for the inhibition of cell proliferation. Phosphorylation of Src/FAK and TGF-ß1-mediated epithelial to mesenchymal transition was also down-regulated in MCF-7 hCD24 shRNA cells. Reduced Src/FAK activity was caused by a decrease in integrin ß1 bound with CD24 and subsequent destabilization of integrin ß1. Our results suggest that down-regulation of Raf/MEK/ERK signaling via Src/FAK may be dependent on integrin ß1 function and that this mechanism is largely responsible for the CD24 ablation-induced decreases in cell proliferation and epithelial to mesenchymal transition.

Induction of apoptotic cell death by phytoestrogens by up-regulating the levels of phospho-p53 and p21 in normal and malignant estrogen receptor α-negative breast cells.

In this study, we investigated the underlying mechanism by which phytoestrogens suppress the growth of normal (MCF-10A) and malignant (MDA-MB-231) estrogen receptor α (ERα)-negative breast cells. We hypothesized that phytoestrogen inhibits the proliferation of ERα-negative breast cancer cells. We found that all tested phytoestrogens (genistein, apigenin, and quercetin) suppressed the growth of both MCF-10A and MDA-MB-231 cells, as revealed by proliferation assays. These results were accompanied by an increase in the sub-G0/G1 apoptotic fractions as well as an increase in the cell population in the G2/M phase in both cell types, as revealed by cell cycle analysis. When we assessed the effect of phytoestrogens on the level of intracellular signaling molecules by Western blot analysis, we found that phytoestrogens increased the level of active p53 (phospho-p53) without changing the p53 level in both MCF-10A and MDA-MB-231 cells. Phytoestrogens also induced an increase in p21, a p53 target gene, and a decrease in either Bcl-xL or cyclin B1 in both cell types. In contrast, the protein levels of phosphatase and tensin homolog, cyclin D1, cell division control protein 2 homolog, phospho-cell division control protein 2 homolog, and p27 were not changed after phytoestrogen treatment. Our data indicate that phytoestrogens induce apoptotic cell death of ERα-negative breast cancer cells via p53-dependent pathway and suggest that phytoestrogens may be promising agents in the treatment and prevention of ERα-negative breast cancer.

PTEN sensitizes MDA-MB-468 cells to inhibition of MEK/Erk signaling for the blockade of cell proliferation.

Phosphatase and tensin homolog (PTEN) is a tumor suppressor that inhibits PI3K/Akt signaling. To examine the effect of PTEN on breast cancer cell proliferation, we expressed PTEN in MDA-MB-468 cells (MDA-MB-468 PTEN) by retroviral infection and tested the cell proliferation rate. We found that the growth rate of MDA-MB-468 PTEN cells was significantly lower than that of MDA-MB-468 control vector cells (MDA-MB-468 vec). When the PI3K/Akt signaling inhibitor LY294002 and the MEK/Erk signaling inhibitor U0126 were applied, LY294002 reduced cell proliferation in MDA-MB-468 PTEN and MDA-MB-468 vec by 20%, while U0126 led to a >60% reduction in MDA-MB-468 PTEN and a 20% reduction in MDA-MB-468 vec cells. FACS analysis demonstrated that the subG0/G1 apoptotic fraction was significantly increased in MDA-MB-468 PTEN cells after U0126 treatment, while LY294002 treatment in both cell lines and U0126 treatment in MDA-MB-468 vec cells led to a modest increase in the apoptotic fraction. This phenomenon was accompanied by the down-regulation of p-Erk. p-Erk levels were significantly lower after U0126 treatment in MDA-MB-468 PTEN cells. Similar results were observed in MDA-MB-231 cells, which express endogenous PTEN. The growth of MDA-MB-231 cells was significantly inhibited after U0126 treatment, compared to LY294002, while PTEN-null ZR-75-1 cells did not show increased sensitivity to U0126 over LY294002. Taken together, these findings suggest that blockade of PI3K/Akt signaling by PTEN may render breast cancer cells more dependent on the MEK/Erk pathway for their proliferation and survival.