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The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins (“aggresomes”) in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer. (Cancer Res 2006; 66(7): 3773-81)

We previously reported that ecto-5′nucleotidase (CD73)-generated adenosine prevents hypoxia-mediated breakdown of the epithelial barrier in normal endometrium in vivo and that CD73 is down-regulated in advanced stage endometrial carcinomas (EC). Loss of CD73 occurred specifically in the carcinoma cells. CD73-generated adenosine is heightened in tissues overwhelmed by inflammation, ischemia, or hypoxia. We hypothesized that the loss of CD73 in EC supports tumor progression by negating the reflex of extracellular adenosine to protect epithelial cell integrity. CD73 was associated with epithelial differentiation in vitro in mesenchymal-to-epithelial transitioning cells. Well-differentiated EC cells showed localization of CD73 to cell-cell contacts. Transmission electron microscopy (TEM) together with enzyme histochemistry revealed CD73 was localized to filopodia at cell-cell contacts. CD73 activity also associated with membrane zippers, a filopodia-based mechanism for forming cell-cell adhesions. CD73 was strongly associated with membrane F-actin. CD73 inhibitor, α, β-methylenediphosphate (AoPCP), reduced total F-actin in hypoxic (1% O2, 5% CO2) EC cells. Phalloidin staining showed AoPCP was specifically reducing membrane F-actin. EC cells expressed two adenosine receptors, A1R and A2BR. Inhibition of A1R by the receptor antagonist, DPCPX, decreased total F-actin. A1R antagonism reduced membrane F-actin. TEM showed A1R antagonism shortened the length of cell-cell filopodia. N(6)-cyclopentyladenosine (CPA), an A1R agonist, increased total F-actin and was membrane specific. This led to the assembly of the actin-related protein 2/3 complex, an actin polymerization complex of filopodia. Rho GTPase, cell division control protein 42 (Cdc42) co-immunoprecipitated with neuronal Wiskott-Aldrich syndrome protein (N-WASP). CPA increased E-cadherin, β-catenin and Na+K+ ATPase in membrane fractions. EC cell migration and invasion was increased by CD73 siRNA and AoPCP and was reversed by 5′-N-ethylcarboxamidoadenosine, an adenosine receptor analog. In summary, we show that CD73-generated adenosine induces an important physiological reflex, inherent to non-neoplastic epithelial cells, to re-form cell-cell adhesions in response to hypoxic stress. Epithelial cells are inherently programmed to maintain cell-cell adhesions, and like other epithelial characteristics this reflex must be down-regulated for tumor progression. Understanding the biology of extracellular adenosine in tumors has the potential to contribute to the discovery of new therapeutic targets, as agonists and antagonists of adenosine receptors are currently being investigated in clinical trials of non-cancer diseases. (NIH 1P50CA098258-01) Citation Format: Jessica L. Bowser, Michael R. Blackburn, Kenneth Dunner, Russell R. Broaddus. CD73 induces actin polymerization to protect epithelial cell-cell adhesions: Loss of this physiological reflex in endometrial carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2015-336

Bortezomib (Velcade, formerly known as PS-341) is a boronic acid dipeptide derivative that is a selective and potent inhibitor of the proteasome. We hypothesized that proteasome inhibition would lead to an accumulation of misfolded proteins in the cell resulting in endoplasmic reticulum (ER) stress. The ability of bortezomib to induce ER stress and the unfolded protein response was investigated in a human pancreatic cancer cell line, L3.6pl. Bortezomib increased expression of ER stress markers, CHOP and BiP, but inhibited PKR-like ER kinase and subsequent phosphorylation of eukaryotic initiation factor 2α (eif2α), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2α phosphorylation, ubiquitylated protein accumulation, and proteotoxicity. (Cancer Res 2005; 65(24): 11510-9)

Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH2-terminal kinase (JNK)–dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca2+ levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin. (Cancer Res 2005; 65(24): 11658-66)