Heat shock protein inhibitors increase the efficacy of measles virotherapy

Abstract

Oncolytic measles virus strains have activity against multiple tumor types and are currently in phase I clinical testing. Induction of the heat shock protein 70 (HSP70) constitutes one of the earliest changes in cellular gene expression following infection with RNA viruses including measles virus, and HSP70 upregulation induced by heat shock has been shown to result in increased measles virus cytotoxicity. HSP90 inhibitors such as geldanamycin (GA) or 17-allylaminogeldanamycin result in pharmacologic upregulation of HSP70 and they are currently in clinical testing as cancer therapeutics. We therefore investigated the hypothesis that heat shock protein inhibitors could augment the measles virus-induced cytopathic effect. We tested the combination of a measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) and GA in MDA-MB-231 (breast), SKOV3.IP (ovarian) and TE671 (rhabdomyosarcoma) cancer cell lines. Optimal synergy was accomplished when GA treatment was initiated 6-24 h following MV infection. Western immunoblotting confirmed HSP70 upregulation in combination-treated cells. Combination treatment resulted in statistically significant increase in syncytia formation as compared to MV-CEA infection alone. Clonogenic assays demonstrated significant decrease in tumor colony formation in MV-CEA/GA combination-treated cells. In addition there was increase in apoptosis by 4,6-diamidino-2-phenylindole staining. Western immunoblotting for caspase-9, caspase-8, caspase-3 and poly(ADP-ribose) polymerase (PARP) demonstrated increase in cleaved caspase-8 and PARP. The pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected tumor cells from MV-CEA/GA-induced PARP activation, indicating that apoptosis in combinationtreated cells occurs mainly via the extrinsic caspase pathway. Treatment of normal cells, such as normal human fibroblasts, however, with the MV-CEA/GA combination, did not result in cytopathic effect, indicating that GA did not alter the MV-CEA specificity for tumor cells. One-step viral growth curves, western immunoblotting for MV-N protein expression, QRT-PCR quantitation of MV-genome copy number and CEA levels showed comparable proliferation of MV-CEA in GAtreated vs -untreated tumor cells. Rho activation assays and western blot for total RhoA, a GTPase associated with the actin cytoskeleton, demonstrated decrease in RhoA activation in combination-treated cells, a change previously shown to be associated with increase in paramyxovirus-induced cell-cell fusion. The enhanced cytopathic effect resulting from measles virus/GA combination supports the translational potential of this approach in the treatment of cancer.

Reduced heat shock protein 70 in airway smooth muscle in patients with chronic obstructive pulmonary disease

“…Moreover, independent of decreased protection in homeostasis of [Ca 2+ ] i , reduced Hsp70 could also increase calcium sensitivity in smooth muscle cells. And reduced Hsp70 could lead to the Ca 2+ sensitivity and smooth muscle contraction increase through a small GTPase protein, RhoA [29][30][31]. Furthermore, some studies demonstrated that overexpression of Hsp70 protected paxillin, a focal adhesion-associated adapter protein, and the dynamic assembly and disassembly of focal adhesions plays a central role in cell mobility [32].…”

Measles to the Rescue: A Review of Oncolytic Measles Virus

“…The expression of Helicobacter pylori neutrophil-activating protein (NAP), by attenuated MV has also been shown to induce a potent anti-tumour immune response in lung and intrapleural metastatic breast cancer xenograft models via stimulating the release of proinflammatory cytokines [77]. MV has also been engineered to code for other immune-stimulating transgenes including interleukin (IL-13) [78] and heat shock protein inhibitors [79]. …”

Potential and clinical translation of oncolytic measles viruses

“…These include: adult acute lymphoblastic leukemia and chronic lymphocytic leukemia [58,59], adult T cell leukemia/lymphoma [60], atypical teratoid rhabdoid tumor [61], breast cancer [44,62], cholangiocarcinoma [63], colorectal cancer [64], cutaneous T cell lymphoma [65], gliomas [66], head and neck squamous cell cancer [67], hepatoblastoma [68], hepatocellular cancer [51], lung cancer [64,69,70], malignant peripheral nerve sheath tumor [71], mantle cell lymphoma [72], medulloblastoma [53,73,74], melanoma [75,76], mesothelioma [77,78], multiple myeloma [47,79], non-Hodgkin’s lymphoma [80], osteosarcoma [81], ovarian cancer [52,82,83], pancreatic cancer [49,50,84], prostate cancer [45,85], renal cell carcinoma [86], rhabdomyosarcoma [87], and splenic marginal zone lymphoma [88]. …”

“…An enhancement of the MV cytopathic effect was seen when the heat shock protein 90 inhibitor geldanamycin, was combined with the oncolytic MV-CEA strain in breast, ovarian and rhabdomyosarcoma cell lines with significant increase in apoptosis mediated via the extrinsic caspase pathway [87]. Inhibition of Rho-associated coiled-coil-forming kinase in combination with MV infection similarly enhanced the cytopathic effect in breast, glioblastoma and prostate cancer cell lines through disruption of the cytoskeleton and increased fusogenicity [123].…”