Efficacy of IGFBP7 for treatment of metastatic melanoma and other cancers in mouse models and human cell lines

We recently identified the secreted protein IGFBP7 as a factor required for an activated BRAF oncogene to induce senescence or apoptosis in primary human cells. In human melanomas containing an activating BRAF mutation (BRAF-positive melanomas), IGFBP7 is epigenetically silenced, which seems to be a critical step in melanoma genesis. Restoration of IGFBP7 function by the addition of recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAF-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses the growth of BRAF-positive primary tumors in xenografted mice. Here we further evaluate the role of IGFBP7 in the treatment of BRAF-positive melanoma and other malignancies. We find that in human metastatic melanoma samples IGFBP7 is epigenetically silenced and at an even higher frequency than that found in primary melanomas. Using a murine experimental metastasis assay, we show that systemic administration of rIGFBP7 markedly suppresses the growth of metastatic disease and prolongs survival. An analysis of the NCI60 panel of human cancer cell lines reveals that in addition to melanoma, IGFBP7 induces apoptosis in several other cancer types, in particular colorectal cancer cell lines. In general, IGFBP7 induces apoptosis in human cancer cell lines that have an activating mutation in BRAF or RAS, and that are sensitive to chemical inhibition of BRAF-MEK-ERK signaling. Significantly, systemically administered rIGFBP7 blocks the growth of colorectal tumors containing an activating RAS or BRAF mutation in mouse xenografts. The results presented here, in conjunction with those from previous studies, justify the further development of IGFBP7 as an anticancer agent

Oncogenic RAS directs silencing of tumor suppressor genes through ordered recruitment of transcriptional repressors

We previously identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). Here we performed RNAi-based epistasis experiments and found that RAS-directed silencing occurs through a highly ordered pathway that is initiated by binding of ZFP354B, a sequence-specific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers

CBX5 Loss Drives EGFR Inhibitor Resistance and Results in Therapeutically Actionable Vulnerabilities in Lung Cancer

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gen

MELK Promotes Melanoma Growth by Stimulating the NF-kappaB Pathway

Melanoma accounts for more than 80% of skin cancer-related deaths, and current therapies provide only short-term benefit to patients. Here, we show in melanoma cells that maternal embryonic leucine zipper kinase (MELK) is transcriptionally upregulated by the MAPK pathway via transcription factor E2F1. MELK knockdown or pharmacological inhibition blocked melanoma growth and enhanced the effectiveness of BRAFV600E inhibitor against melanoma cells. To identify mediators of MELK function, we performed stable isotope labeling with amino acids in cell culture (SILAC) and identified 469 proteins that had downregulated phosphorylation after MELK inhibition. Of these proteins, 139 were previously reported as substrates of BRAF or MEK, demonstrating that MELK is an important downstream mediator of the MAPK pathway. Furthermore, we show that MELK promotes melanoma growth by activating NF-kappaB pathway activity via Sequestosome 1 (SQSTM1/p62). Altogether, these results underpin an important role for MELK in melanoma growth downstream of the MAPK pathway

Oncogenic EGFR Represses the TET1 DNA Demethylase to Induce Silencing of Tumor Suppressors in Cancer Cells

SummaryOncogene-induced DNA methylation-mediated transcriptional silencing of tumor suppressors frequently occurs in cancer, but the mechanism and functional role of this silencing in oncogenesis are not fully understood. Here, we show that oncogenic epidermal growth factor receptor (EGFR) induces silencing of multiple unrelated tumor suppressors in lung adenocarcinomas and glioblastomas by inhibiting the DNA demethylase TET oncogene family member 1 (TET1) via the C/EBPα transcription factor. After oncogenic EGFR inhibition, TET1 binds to tumor suppressor promoters and induces their re-expression through active DNA demethylation. Ectopic expression of TET1 potently inhibits lung and glioblastoma tumor growth, and TET1 knockdown confers resistance to EGFR inhibitors in lung cancer cells. Lung cancer samples exhibited reduced TET1 expression or TET1 cytoplasmic localization in the majority of cases. Collectively, these results identify a conserved pathway of oncogenic EGFR-induced DNA methylation-mediated transcriptional silencing of tumor suppressors that may have therapeutic benefits for oncogenic EGFR-mediated lung cancers and glioblastomas

Synergistic tumor suppression by combined inhibition of telomerase and CDKN1A

Tumor suppressor p53 plays an important role in mediating growth inhibition upon telomere dysfunction. Here, we show that loss of the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21WAF1/CIP1) increases apoptosis induction following telomerase inhibition in a variety of cancer cell lines and mouse xenografts. This effect is highly specific to p21, as loss of other checkpoint proteins and CDK inhibitors did not affect apoptosis. In telomerase, inhibited cell loss of p21 leads to E2F1- and p53-mediated transcriptional activation of p53-upregulated modulator of apoptosis, resulting in increased apoptosis. Combined genetic or pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simultaneous inhibition of telomerase and p21 also suppresses growth of tumors containing mutant p53 following pharmacological restoration of p53 activity. Collectively, our results establish that inactivation of p21 leads to increased apoptosis upon telomerase inhibition and thus identify a genetic vulnerability that can be exploited to treat many human cancers containing either wild-type or mutant p53

Cancer genomics and proteomics : methods and protocols / edited by Narendra Wajapeyee.

pharmacy bookfair2015Includes bibliographical references and index.xi, 262 pages

Pharmacogenomics in breast cancer: current trends and future directions

Pharmacogenomics is the study of genetic variations between individuals to predict the risk of toxic side effects and the probability that a patient will respond to single- or multidrug chemotherapy. Breast cancer remains one of the most common cancers among women worldwide and is second only to lung cancer in cancerrelated death. A better understanding of the mechanisms of initiation and progression of breast cancer is needed for early diagnosis and development of better therapeutic methodologies. Differences in cancer patients' responses to chemotherapy have often been attributed to pathogenesis and severity of the disease, drug interactions, patient's age, gender, nutritional status, organ functions and tumor biology. It is now well recognized that genetic variations in drug target genes, disease pathway genes and drug metabolizing enzymes can have greater influence on drug efficacy and toxicity. In addition, germline variants can be used to study breast cancer susceptibility, as well as the variable response to both drug and radiation therapy used in the treatment of breast cancer. This review discusses clinically relevant individual gene variations that influence breast cancer susceptibility and cancer therapy, as well as the microarray-based expression profiling studies that have great potential in cancer pharmacogenomics in terms of tumor classification, drug and biomarker discovery and drug efficacy testing

Cell Cycle Arrest and Apoptosis Induction by Activator Protein 2\alpha (AP-2\alpha) and the Role of p53p53 and p21WAF1/CIP1p21^{WAF1/CIP1} in AP-2\alpha-mediated Growth Inhibition

Activator protein 2\alpha (AP-2\alpha) is a sequence-specific DNA-binding transcription factor implicated in differentiation and transformation. In this study, we have made a replication-deficient recombinant adenovirus that expresses functional AP-2\alpha(Ad-AP2). Cells infected with Ad-AP2 expressed induced levels of AP-2\alpha protein, which bound to DNA in a sequence-specific manner and activated the AP-2-specific reporter 3X-AP2. Expression of AP-2\alpha from Ad-AP2 inhibited cellular DNA synthesis and induced apoptosis. Ad-AP2 infection resulted in efficient inhibition of growth of cancer cells of six different types. In addition, prior expression of AP-2\alpha increased the chemosensitivity of H460, a lung carcinoma cell line, to adriamycin (2.5-fold) and cisplatin (5-fold). Furthermore, the growth inhibition by AP-2\alpha was found to be less efficient in the absence of p53 or p21, which correlated with reduced apoptosis in p53 null cells and lack of DNA synthesis inhibition in $p21^{WAF1/CIP1}$ null cells by AP-2\alpha, respectively. These results suggest that AP-2\alpha inhibits the growth of cells by inducing cell cycle arrest and apoptosis and that the use of AP-2\alpha should be explored as a therapeutic strategy either alone or in combination with chemotherapy

Role of p53 status in chemosensitivity determination of cancer cells against histone deacetylase inhibitor sodium butyrate

Histone deacetylases inhibitors (HDIs) induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (NaB), a histone deacetylase inhibitor, has been shown to cause a G(1) cell cycle arrest by inducing p21(WAF1/CIP1) in a p53-independent manner. In this report, we present evidence for activation of p53 pathway by NaBand its role in the NaB-mediated growth suppression. Addition of NaB increased the levels of p53 involving a p14(ARF)-dependent post-transcriptional mechanism. NaB induced p53 is functional as it activated p53-specific reporter, induced the level of p21(WAF1/CIP1),inhibited cellular DNA synthesis and induced apoptosis. By using HPV 16E6 stable transfectants as well as p53 null cancer cells, we show that NaB suppresses the growth of WT p53 containing cells more efficiently.NaB inhibited DNA synthesis to similar extent both in the presence and absence of p53. However, NaB treatment lead to a major G(2)/M arrest of cells in the presence of p53, while cells without wild-type p53 accumulated mainly in G, phase of the cell cycle. Furthermore,apoptosis induction by NaB is greatly reduced in the absence of p53.These results suggest that p53 pathway mediates in part growth suppression by NaB and the p53 status may be an important determinant of chemosensitivity in HDI based cancer chemotherapy