Effect of inhibition of the Ubiquitin-Proteasome System and Hsp90 on growth and survival of Rhabdomyosarcoma cells in vitro

Abstract

BackgroundThe ubiquitin-proteasome system (UPS) and the heat shock response (HSR) are two critical regulators of cell homeostasis, as their inhibition affects growth and survival of normal cells, as well as stress response and invasiveness of cancer cells. We evaluated the effects of the proteasome inhibitor Bortezomib and of 17-DMAG, a competitive inhibitor of Hsp90, in rhabdomyosarcoma (RMS) cells, and analyzed the efficacy of single-agent exposures with combination treatments.MethodsTo assess cytotoxicity induced by Bortezomib and 17-DMAG in RMS cells, viability was measured by MTT assay after 24, 48 and 72 hours. Western blotting and immunofluorescence analyses were carried out to elucidate the mechanisms of action. Apoptosis was measured by FACS with Annexin-V-FITC and Propidium Iodide.ResultsBortezomib and 17-DMAG, when combined at single low-toxic concentrations, enhanced growth inhibition of RMS cells, with signs of autophagy that included intensive cytoplasmic vacuolization and conversion of cytosolic LC3-I protein to its autophagosome-associated form. Treatment with lysosomal inhibitor chloroquine facilitates apoptosis, whereas stimulation of autophagy by rapamycin prevents LC3-I conversion and cell death, suggesting that autophagy is a resistance mechanism in RMS cells exposed to proteotoxic drugs. However, combination treatment also causes caspase-dependent apoptosis, PARP cleavage and Annexin V staining, as simultaneous inhibition of both UPS and HSR systems limits cytoprotective autophagy, exacerbating stress resulting from accumulation of misfolded proteins.ConclusionThe combination of proteasome inhibitor Bortezomib with Hsp90 inhibitor 17-DMAG, appears to have important therapeutic advantages in the treatment of RMS cells compared with single-agent exposure, because compensatory survival mechanisms that occur as side effects of treatment may be prevented.

Compensatory increases of select proteostasis networks after Hsp70 inhibition in cancer cells

“…This conclusion is supported by the findings showing increased levels of the mRNAs and proteins linked to autophagosome elongation, and LC3BII conjugation to the autophagosomal membrane as well as by electron microscopy. The pro-survival activity of the autophagy pathway in select cancers has been established (Clarke et al, 2012;Hambright and Ghosh, 2017;Jiang and Mizushima, 2014;Levine and Kroemer, 2008;Maycotte and Thorburn, 2014), and, consistent with our results, rhabdomyosarcomas also coopt this pro-survival pathway (Peron et al, 2012;Rapino et al, 2014). Surprisingly, inhibition of autophagy, but not ERAD, re-sensitized RMS13-R cells to MAL3-101.…”

Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells

“…This pilot study has expanded our understanding of the cross-talk between apoptosis and autophagy controlling TMZ-induced apoptosis. It has been shown that the maintenance of cellular viability in RH30 cells is dependent on baseline autophagy 25 , while loss of ATG7 or Baf-A1 treatment can negatively regulate proliferation of RH30 cells 26 , 27 . We found that Baf-A1 inhibited baseline autophagy in RH30 and C2C12 cells and decreased their viability.…”

“…In RH30 cells, autophagy is known to be a crucial process in the maintenance of cellular viability and proliferation 25 . Furthermore, inhibition of autophagy by the Atg7 knockdown, or pharmacological inhibition with chloroquine or Baf-A1 treatment, has been demonstrated to decrease cell growth and reduced viability in RMS cell lines 26 , 27 .…”

Identification of clinically achievable combination therapies in childhood rhabdomyosarcoma

“…The proteasome inhibitor, bortezomib, functions through inhibition of the 26S proteasome, leading to apoptosis, cell cycle arrest, and deregulated NF-KB signaling and is currently approved for use in hematologic malignancies [ 17 – 20 ]. In vitro studies have shown RMS cell lines to exhibit increased rates of apoptosis and cell cycle arrest when treated with bortezomib both as a single agent as well as in combination [ 19 , 20 ]. AZD1775 is a selective tyrosine kinase inhibitor of the Wee1 kinase which regulates the cell cycle through phosphorylation and inhibition of cyclin-dependent kinase 1.…”