MUC1 Is a Downstream Target of STAT3 and Regulates Lung Cancer Cell Survival and Invasion

Signal transducer and activator of transcription 3 (STAT3) is aberrantly activated in human cancer including lung cancer and has been implicated in transformation, tumorigenicity, and metastasis. One putative downstream gene regulated by Stat3 is MUC1 which also has important roles in tumorigenesis. We determined if Stat3 regulates MUC1 in lung cancer cell lines and what function MUC1 plays in lung cancer cell biology. We examined MUC1 expression in non-small cell lung cancer (NSCLC) cell lines and found high levels of MUC1 protein expression associated with higher levels of tyrosine phosphorylated STAT3. STAT3 knockdown downregulated MUC1 expression whereas constitutive STAT3 expression increased MUC1 expression at mRNA and protein levels. MUC1 knockdown induced cellular apoptosis concomitant with reduced Bcl-XL and sensitized cells to cisplatin treatment. MUC1 knockdown inhibited tumor growth and metastasis in an orthotopic mouse model of lung cancer by activating apoptosis and inhibiting cell proliferation in vivo. These results demonstrate that constitutively activated STAT3 regulates expression of MUC1, which mediates lung cancer cell survival and metastasis in vitro and in vivo. MUC1 appears to be a cooperating oncoprotein with multiple oncogenic tyrosine kinase pathways and could be an effective target for the treatment of lung cancer

Histone Deacetylase Inhibitors Downregulate Checkpoint Kinase 1 Expression to Induce Cell Death in Non-Small Cell Lung Cancer Cells

Background: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods and Findings: Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACiinduced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. Conclusions: These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples

Heregulin-stimulated acetylcholine receptor gene expression in muscle: requirement for MAP kinase and evidence for a parallel inhibitory pathway independent of electrical activity

tion of the of their intrinsic tyrosine kinases, initiating a cascade two receptors (Sliwkowski et al., 1994; Alimandi et al., of events resulting in the stimulation of acetylcholine 1995; Altiok et al., 1995; Marikovsky et al., 1995). receptor (AChR) genes in muscle. Here we have Activation of receptor tyrosine kinases creates binding examined the signalling downstream of the HRG recep- sites for intracellular signalling proteins containing src tor. We show that phosphatidylinositol 39-kinase (PI3K) homology 2 (SH2) domains, including those with enzym- and SHC bind to the HRG-activated ErbB3 in atic activity such as phospholipase C-g and phosphatidyl- myotubes. Subsequently, p70S6 kinase (p70 S6k ), and inositol 39-kinase (PI3K), and those with no

JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells

Estrogen is known to stimulate breast cancer development in humans. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The mechanism by which estrogen induces tumour regression in breast cancer is still unknown. We found that under low growth-stimulated conditions, high concentrations of 17-beta-estradiol (estradiol) induces apoptosis and concomitantly increases phosphorylation of c-jun in estrogen receptor (ER)-positive breast cancer cell line, MCF-7, but not in ER-negative breast cancer cell line MDA-MB 231 suggesting an ER-mediated event. Interestingly, when the c-jun NH2-terminal kinase (JNK) signalling pathway was disrupted by the JNK inhibitor SP600125, the ability of estradiol to inhibit the growth of MCF-7 cells and to induce apoptosis was completely blocked. These data suggest that JNK plays a central role in mediating the anticancer effect of high concentrations of estradiol in MCF-7 cells. Our data showing the apoptotic effect of estradiol in low growth-stimulated conditions suggest potential implications for the pharmacological control of breast cancer with high dose estrogen in postmenopausal women. Furthermore, our results indicate that augmenting JNK activity could be an efficient novel approach for treating breast cancer

A plant oxylipin, 12-oxo-phytodienoic acid, inhibits proliferation of human breast cancer cells by targeting cyclin D1

Cyclin D1 overexpression has been associated with poor prognosis and resistance to therapy in human breast cancer. Thus, the development of therapeutic agents that selectively target cyclin D1 activity is of clinical interest. This study demonstrates that 12-oxo-phytodienoic acid (OPDA), a phytohormone with critical functions in growth and development in plants, induces growth arrest in MDA-MB-231 and T47D breast cancer cells. In response to OPDA treatment, the human breast cancer cell lines exhibit a progressive decline in cyclin D1 expression, which is tightly associated with the accumulation of hypophosphorylated form of the retinoblastoma protein (Rb) and G1 arrest. The decrease in cyclin D1 protein expression accompanies a dramatic decline in nuclear but not membranous beta-catenin expression and activation of glycogen synthase kinase-3-beta (GSK3 beta) caused by inhibition of its serine-9 phosphorylation. The proteasome inhibitor MG132 blocks OPDA-mediated decrease in cyclin D1. In addition, the overexpression of T286A, a cyclin D1 mutant which is refractory to phosphorylation by GSK3b and proteosomal degradation, is resistant to OPDA-mediated Rb dephosphorylation as well as G1 cell cycle arrest. Thus, our results demonstrate that degradation of cyclin D1 protein is a key event in OPDA induced growth inhibition in breast cancer cells. These data provide the basic foundation for future efforts to develop OPDA-based approaches in the prevention and treatment of breast cancer and other types of cancer

Cancer Control 193 The Old Barn. Photograph courtesy of Craig Damlo

with erlotinib, carboplatin, paclitaxel, and bevacizumab in addition to radiation and consolidation therapy with erlotinib and bevacizumab have objective response rates between 39% and 60% and median progression-free survival (PFS) of 10.2 months to 18.4 months. 2 Response rates for patients with metastatic NSCLC provided treatment are 17% to 37%; median survival rates are 6.7 to 11.3 months; and 1-and 2-year survival rates are 31% to 46% and 9% to 21%, respectively. 1,3-5 Time to progression averages 4 to 6 months and response rate to second-line therapy is 8%. EGFR and Tyrosine Kinase Inhibitors The most promising and widely reported molecular predictive factor in NSCLC is EGFR, which resides on Survival and Response Rates Across all treatments and tumor stages, the 5-year overall survival (OS) rate for non-small-cell lung cancer (NSCLC) is 16%. 1 Patients receiving induction chemotherapy with carboplatin, paclitaxel, and bevacizumab followed by concurrent chemotherapy Advances in EGFR as a Predictive Marker in Lung Adenocarcinom

EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer

Abstract Background Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling. Results SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples. Conclusions Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.</p

Microfluidic Biopsy Trapping Device for the Real-Time Monitoring of Tumor Microenvironment

<div><p>The tumor microenvironment is composed of cellular and stromal components such as tumor cells, mesenchymal cells, immune cells, cancer associated fibroblasts and the supporting extracellular matrix. The tumor microenvironment provides crucial support for growth and progression of tumor cells and affects tumor response to therapeutic interventions. To better understand tumor biology and to develop effective cancer therapeutic agents it is important to develop preclinical platforms that can faithfully recapitulate the tumor microenvironment and the complex interaction between the tumor and its surrounding stromal elements. Drug studies performed in vitro with conventional two-dimensional cancer cell line models do not optimally represent clinical drug response as they lack true tumor heterogeneity and are often performed in static culture conditions lacking stromal tumor components that significantly influence the metabolic activity and proliferation of cells. Recent microfluidic approaches aim to overcome such obstacles with the use of cell lines derived in artificial three-dimensional supportive gels or micro-chambers. However, absence of a true tumor microenvironment and full interstitial flow, leads to less than optimal evaluation of tumor response to drug treatment. Here we report a continuous perfusion microfluidic device coupled with microscopy and image analysis for the assessment of drug effects on intact fresh tumor tissue. We have demonstrated that fine needle aspirate biopsies obtained from patient-derived xenograft models of adenocarcinoma of the lung can successfully be analyzed for their response to ex vivo drug treatment within this biopsy trapping microfluidic device, wherein a protein kinase C inhibitor, staurosporine, was used to assess tumor cell death as a proof of principle. This approach has the potential to study tumor tissue within its intact microenvironment to better understand tumor response to drug treatments and eventually to choose the most effective drug and drug combination for individual patients in a cost effective and timely manner.</p></div