Citations

ABSTRACT Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aβ peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aβ1‐42 oligomers can readily enter a senescence phenotype. To determine the effect of Aβ oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aβ1‐42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aβ1‐42 monomers, and 5 µM Aβ1‐42 fibrils, respectively. Brain ECs treated with Aβ1‐42 oligomers showed increased senescence‐associated β‐galactosidase staining and increased senescence‐associated p21/ p53 expression. Treatment with either Aβ1‐42 monomer or Aβ1‐42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aβ1‐42 oligomer‐treated ECs, and these cells showed significantly increased VEGFR‐1 expression and decreased VEGFR‐2 levels. Overexpression of VEGFR‐1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR‐1 signaling events in this paradigm. More importantly, small interfering RNA‐mediated knockdown of VEGFR‐1 expression in brain ECs was able to prevent up‐regulation of p21 protein expression and significantly reduced induction of senescence following Aβ1‐42 oligomer treatment. Our studies show that exposure to Aβ1‐42 oligomers may impair vascular functions by altering VEGFR‐1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR‐1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD.—Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor‐1 modulates amyloid β 1–42 oligomer‐induced senescence in brain endothelial cells. FASEB J. 33, 4626–4637 (2019). www.fasebj.org

Tumor-selective uptake of plectin-1 targeting peptide-modified gold nanoparticles ameliorates targeted delivery of gemcitabine in pancreatic ductal adenocarcinoma.

603 Background: Oncogenic BRAFV600E mutations activate MAP kinase signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer (CRC). In BRAFV600E mutant CRCs, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. Methods: BRAF isogenic RKO CRC cells and RKO, HT29, WiDr cell lines were treated with cobimetinib ± the small molecule MCL-1 inhibitor, A-1210477. Apoptosis was measured by Annexin V staining and caspase cleavage. Gene knockdown or overexpression was achieved by lentivirus; ERK siRNA was utilized. Competitive RT-PCR was performed for MCL-1 mRNA expression. HT-29 cells with control or MCL-1 shRNA were xenografted into SCID mice, treated with cobimetinib or vehicle, and tumor volume was measured. Results: We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using CRC cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by ERK siRNA that suppressed MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively.MEK /ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced pro-apoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown vs control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. A-1210477 also enhanced cobimetinib-induced apoptosis in vitrothat was due to disruption of the interaction of MCL-1 with pro-apoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. Conclusions: BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism.

Abstract Oncogenic BRAFV600E mutations activate MAPK signaling and are associated with treatment resistance and poor prognosis in patients with colorectal cancer. In BRAFV600E-mutant colorectal cancers, treatment failure may be related to BRAFV600E-mediated apoptosis resistance that occurs by an as yet undefined mechanism. We found that BRAFV600E can upregulate anti-apoptotic MCL-1 in a gene dose-dependent manner using colorectal cancer cell lines isogenic for BRAF. BRAFV600E-induced MCL-1 upregulation was confirmed by ectopic BRAFV600E expression that activated MEK/ERK signaling to phosphorylate (MCL-1Thr163) and stabilize MCL-1. Upregulation of MCL-1 was mediated by MEK/ERK shown by the ability of ERK siRNA to suppress MCL-1. Stabilization of MCL-1 by phosphorylation was shown by a phosphorylation-mimicking mutant and an unphosphorylated MCL-1 mutant that decreased or increased MCL-1 protein turnover, respectively. MEK/ERK inhibition by cobimetinib suppressed MCL-1 expression/phosphorylation and induced proapoptotic BIM to a greater extent than did vemurafenib in BRAFV600E cell lines. MCL-1 knockdown versus control shRNA significantly enhanced cobimetinib-induced apoptosis in vitro and in HT29 colon cancer xenografts. The small-molecule MCL-1 inhibitor, A-1210477, also enhanced cobimetinib-induced apoptosis in vitro that was due to disruption of the interaction of MCL-1 with proapoptotic BAK and BIM. Knockdown of BIM attenuated BAX, but not BAK, activation by cobimetinib plus A-1210477. In summary, BRAFV600E-mediated MEK/ERK activation can upregulate MCL-1 by phosphorylation/stabilization to confer apoptosis resistance that can be reversed by MCL-1 antagonism combined with cobimetinib, suggesting a novel therapeutic strategy against BRAFV600E-mutant CRCs. Mol Cancer Ther; 15(12); 3015–27. ©2016 AACR.

Abstract Tumor metastasis refers to spread of a tumor from site of its origin to distant organs and causes majority of cancer deaths. Although >30 metastasis suppressor genes (MSGs) that negatively regulate metastasis have been identified so far, two issues are poorly understood: first, which MSGs oppose metastasis in a tumor type, and second, which molecular function of MSG controls metastasis. Herein, integrative analyses of tumor-transcriptomes (n = 382), survival data (n = 530) and lymph node metastases (n = 100) in lung cancer patients identified non-metastatic 2 (NME2) as a key MSG from a pool of >30 metastasis suppressors. Subsequently, we generated a promoter-wide binding map for NME2 using chromatin immunoprecipitation with promoter microarrays (ChIP-chip), and transcriptome profiling. We discovered novel targets of NME2 which are involved in focal adhesion signaling. Importantly, we detected binding of NME2 in promoter of focal adhesion factor, vinculin. Reduced expression of NME2 led to enhanced transcription of vinculin. In comparison, NME1, a close homolog of NME2, did not bind to vinculin promoter nor regulate its expression. In line, enhanced metastasis of NME2-depleted lung cancer cells was found in zebrafish and nude mice tumor models. The metastatic potential of NME2-depleted cells was remarkably diminished upon selective RNA-i-mediated silencing of vinculin. Together, we demonstrate that reduced NME2 levels lead to transcriptional de-repression of vinculin and regulate lung cancer metastasis.

Abstract Endoglin, a 180-kDa disulfide-linked homodimeric transmembrane receptor protein mostly expressed in tumor-associated endothelial cells, is an endogenous binding partner of GAIP-interacting protein, C terminus (GIPC). Endoglin functions as a coreceptor of TβRII that binds TGFβ and is important for vascular development, and consequently has become a compelling target for antiangiogenic therapies. A few recent studies in gastrointestinal stromal tumor (GIST), breast cancer, and ovarian cancer, however, suggest that endoglin is upregulated in tumor cells and is associated with poor prognosis. These findings indicate a broader role of endoglin in tumor biology, beyond angiogenic effects. The goal of our current study is to evaluate the effects of targeting endoglin in pancreatic cancer both in vitro and in vivo. We analyzed the antiproliferative effect of both RNAi-based and peptide ligand-based inhibition of endoglin in pancreatic cancer cell lines, the latter yielding a GIPC PDZ domain-targeting lipopeptide with notable antiproliferative activity. We further demonstrated that endoglin inhibition induced a differentiation phenotype in the pancreatic cancer cells and sensitized them against conventional chemotherapeutic drug gemcitabine. Most importantly, we have demonstrated the antitumor effect of both RNAi-based and competitive inhibitor–based blocking of endoglin in pancreatic cancer xenograft models in vivo. To our knowledge, this is the first report exploring the effect of targeting endoglin in pancreatic cancer cells. Mol Cancer Ther; 13(10); 2264–75. ©2014 AACR.

Abstract Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase, is a key regulator of numerous cellular processes ranging from glycogen metabolism to cell-cycle regulation and proliferation. Consistent with its involvement in many pathways, it has also been implicated in the pathogenesis of various human diseases, including type II diabetes, Alzheimer disease, bipolar disorder, inflammation, and cancer. Consequently, it is recognized as an attractive target for the development of new drugs. In the present study, we investigated the effect of both pharmacologic and genetic inhibition of GSK-3 in two different renal cancer cell lines. We have shown potent antiproliferative activity of 9-ING-41, a maleimide-based GSK-3 inhibitor. The antiproliferative activity is most likely caused by G0–G1 and G2–M phase arrest as evident from cell-cycle analysis. We have established that inhibition of GSK-3 imparted a differentiated phenotype in renal cancer cells. We have also shown that GSK-3 inhibition induced autophagy, likely as a result of imbalanced energy homeostasis caused by impaired glucose metabolism. In addition, we have demonstrated the antitumor activity of 9-ING-41 in two different subcutaneous xenograft renal cell carcinoma tumor models. To our knowledge, this is the first report describing autophagy induction due to GSK-3 inhibition in renal cancer cells. Mol Cancer Ther; 13(2); 285–96. ©2013 AACR.

Abstract VEGF/vascular permeability factor (VEGF/VPF or VEGF-A) is a pivotal driver of cancer angiogenesis that is a central therapeutic target in the treatment of malignancy. However, little work has been devoted to investigating functions of VEGF that are independent of its proangiogenic activity. Here, we report that VEGF produced by tumor cells acts in an autocrine manner to promote cell growth through interaction with the VEGF receptor neuropilin-1 (NRP-1). Reducing VEGF expression by tumor cells induced a differentiated phenotype in vitro and inhibited tumor forming capacity in vivo, independent of effects on angiogenesis. Autocrine activation of tumor cell growth was dependent on signaling through NRP-1, and Ras was determined to be a critical effector signaling molecule downstream of NRP-1. Our findings define a novel function for VEGF in dedifferentiation of tumor cells expanding its role in cancer beyond its known proangiogenic function. Cancer Res; 72(16); 3912–8. ©2012 AACR.