The Effect of Endothelin-1 on the expression of CDK Inhibitors p21 & p27 in Bovine Corneal Endothelial Cells

Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell proliferation. Confluent serum starved cells grown in T-25 flasks were treated with 100nM Endothelin-1 in DMEM. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of p21cip1 and p27kip1 proteins relative to β-actin was determined by western blotting technique. Immuno fluorescent localization of p27kip1 was performed using polyclonal anti-p27kip1 and anti-p21cip1 antibodies in confluent and growing cells. An increase in cell proliferation was observed in sub-confluent cultures with Endothelin-1 treatment. This evidence was supported by an increase (~18%) in BrdU incorporation in response to Endothelin-1. Densitometry analysis of immunoblots revealed an increase in the expression of p27kip1 in confluent cell cultures when compared to sub-confluent, dividing cells. p21cip1 was almost undetectable in sub-confluent, actively dividing cultures. Immuno fluorescent analysis revealed that the nuclear staining of p27kip1 was apparently decreased with ET-1 treatment. In conclusion, Endothelin-1 treatment resulted in decrease in p27kip1 and p21cip1 expression in confluent cultures that was greatest at 30 hr of post incubation with Endothelin-1. Endothelin-1 appears to promote cell proliferation. Expression of p27kip1 and p21cip1 was greatly reduced in actively dividing BCECs. Endothelin-1 treatment down-regulated these cyclin dependent kinase inhibitors and may promote cell cycle progression via this mechanism

Intracellular activation of EGFR by fatty acid synthase dependent palmitoylation

Epidermal growth factor receptor (EGFR) is an oncogenic receptor tyrosine kinase. Canonically, the tyrosine kinase activity of EGFR is regulated by its extracellular ligands. However, ligand-independent activation of EGFR exists in certain cancer cells, and the underlying mechanism remains to be defined. In this study, using PC3 and A549 cells as a model, we have found that, in the absence of extracellular ligands, a subpopulation of EGFR is constitutively active, which is needed for maintaining cell proliferation. Furthermore, we have found that fatty acid synthase (FASN)-dependent palmitoylation of EGFR is required for EGFR dimerization and kinase activation. Inhibition of FASN or palmitoyl acyltransferases reduced the activity and down-regulated the levels of EGFR, and sensitized cancer cells to EGFR tyrosine kinase inhibitors. It is concluded that EGFR can be activated intracellularly by FASN-dependent palmitoylation. This mechanism may serve as a new target for improving EGFR-based cancer therapy

Alcohol Regulates Genes that Are Associated with Response to Endocrine Therapy and Attenuates the Actions of Tamoxifen in Breast Cancer Cells

Hereditary, hormonal, and behavioral factors contribute to the development of breast cancer. Alcohol consumption is a modifiable behavior that is linked to increased breast cancer risks and is associated with the development of hormone-dependent breast cancers as well as disease progression and recurrence following endocrine treatment. In this study we examined the molecular mechanisms of action of alcohol by applying molecular, genetic, and genomic approaches in characterizing its effects on estrogen receptor (ER)-positive breast cancer cells. Treatments with alcohol promoted cell proliferation, increased growth factor signaling, and up-regulated the transcription of the ER target gene GREB1 but not the canonical target TFF1/pS2. Microarray analysis following alcohol treatment identified a large number of alcohol-responsive genes, including those which function in apoptotic and cell proliferation pathways. Furthermore, expression profiles of the responsive gene sets in tumors were strongly associated with clinical outcomes in patients who received endocrine therapy. Correspondingly, alcohol treatment attenuated the anti-proliferative effects of the endocrine therapeutic drug tamoxifen in ER-positive breast cancer cells. To determine the contribution and functions of responsive genes, their differential expression in tumors were assessed between outcome groups. The proto-oncogene BRAF was identified as a novel alcohol- and estrogen-induced gene that showed higher expression in patients with poor outcomes. Knock-down of BRAF, moreover, prevented the proliferation of breast cancer cells. These findings not only highlight the mechanistic basis of the effects of alcohol on breast cancer cells and increased risks for disease incidents and recurrence, but may facilitate the discovery and characterization of novel oncogenic pathways and markers in breast cancer research and therapeutics

Role of De Novo Fatty Acid Synthesis in Intrinsic Activation of EGFR in Cancer

De novo fatty acid synthesis is one of the major upregulated metabolic pathways in cancer. De novo fatty acid synthesis pathway is crucially involved in cell proliferation, survival and drug resistance of cancer cells. This study focused on the role of fatty acid synthesis in intrinsic activation of EGFR and drug resistance in cancer. EGFR is a receptor tyrosine kinase expressed in most human cancers of epithelial origin and advanced cancers. Besides the plasma membranous (pmEGFR) and nuclear localization, EGFR can also exist in mitochondria (mtEGFR). Our studies revealed that EGFR exists in the inner mitochondrial membrane of prostate and breast cancer cells and promotes mitochondrial fusion through increasing the protein levels of PHB2 and OPA1. Interestingly, in this study, we found that activated pmEGFR activates mtEGFR through de novo synthesized palmitate, through palmitoylation, and promotes mitochondrial fusion. Along with mtEGFR, we also found that pmEGFR also undergo de novo fatty acid synthesis dependent palmitoylation which is important for EGFR localization, stability and signaling. We also observed intrinsic activation of EGFR in response to anticancer drugs such as SN38 and oxaliplatin dependent on de novo fatty acid synthesis. Finally, targeting de novo fatty acid synthesis or palmitoylation significantly increased the sensitivity of cancer cells to EGFR TKIs or anticancer drugs. In conclusion, through this study we’ve uncovered the importance of de novo fatty acid synthesis in intrinsic activation and non-classical functions of EGFR in cancer.Biology and Biochemistry, Department o

Molecular Pathways: Targeting Protein Tyrosine Phosphatases in Cancer

The aberrant activation of oncogenic signaling pathways is a universal phenomenon in cancer and drives tumorigenesis and malignant transformation. This abnormal activation of signaling pathways in cancer is due to the altered expression of protein kinases and phosphatases. In response to extracellular signals, protein kinases activate downstream signaling pathways through a series of protein phosphorylation events, ultimately producing a signal response. Protein tyrosine phosphatases (PTP) are a family of enzymes that hydrolytically remove phosphate groups from proteins. Initially, PTPs were shown to act as tumor suppressor genes by terminating signal responses through the dephosphorylation of oncogenic kinases. More recently, it has become clear that several PTPs overexpressed in human cancers do not suppress tumor growth; instead, they positively regulate signaling pathways and promote tumor development and progression. In this review, we discuss both types of PTPs: those that have tumor suppressor activities as well as those that act as oncogenes. We also discuss the potential of PTP inhibitors for cancer therapy. Clin Cancer Res; 23(9); 2136-42. ©2017 AACR

SOX9 Is Essential for Triple-Negative Breast Cancer Cell Survival and Metastasis

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial-mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. IMPLICATIONS: Our studies identified the SOX9 protein as a "master regulator" of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer