The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133high/CD44high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133low/CD44low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133high/CD44high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

Overexpression of heat shock protein 90 (HSP90) is associated with increased tumor cell survival and radioresistance. In this study we explored the efficacy of the novel HSP90 inhibitor AT13387 and examined its radiosensitizing effects in combination with gamma-radiation in 2D and 3D structures as well as mice-xenografts. AT13387 induced effective cytotoxic activity and radiosensitized cancer cells in monolayer and tumor spheroid models, where low drug doses triggered significant synergistic effects on cell survival together with radiation. Furthermore, AT13387 treatment resulted in G2/M-phase arrest and significantly reduced the migration capacity. The expression of selected client proteins involved in DNA repair, cell-signaling and cell growth was downregulated in vitro, though the expression of most investigated proteins recurred after 8–24 h. These results were confirmed in vivo where AT13387 treated tumors displayed effective downregulation of HSP90 and its oncogenic client proteins.In conclusion, our results demonstrate that AT13387 is a potent new cancer drug and effective radiosensitizer in vitro with an excellent in vivo efficacy. AT13387 treatment has the potential to improve external beam therapy and radionuclide therapy outcomes and restore treatment efficacy in cancers that are resistant to initial therapeutic regimes.

Radiotherapy amplifies p53 expression in cancer cells with wild-type (wt) p53. Blocking the negative regulators MDM2 and MDMX stabilizes p53 and may therefore potentiate radiotherapy outcomes. In this study, we investigate the efficacy of the novel anti-MDM2/X stapled peptide PM2 alone and in combination with external gamma radiation and PM2 therapy combined with radiotherapy elicited synergistic therapeutic effects compared with monotherapy in cells with wt p53 in both and assays, whereas these effects did not manifest in p53 cells. Biodistribution and autoradiography ofI-PM2 revealed high and retained uptake homogenously distributed throughout the tumor. In mice carrying wt p53 tumors, PM2 combined with radiotherapy significantly prolonged the median survival by 50%, whereas effects of PM2 therapy on mutant and p53 tumors were negligible. PM2-dependent stabilization of p53 was confirmed with immunohistochemistry. These data demonstrate the potential of the stapled peptide PM2 as a radiotherapy potentiator and suggest that clinical application of PM2 with radiotherapy in wt p53 cancers might improve tumor control. These findings contribute advances to cancer radiotherapy by using novel p53-reactivating stapled peptides as radiosensitizers in wild-type p53 cancers. .

177Lu-DOTATATE was recently approved for the treatment of somatostatin receptor (SSTR)-positive neuroen-docrine tumors (NETs). However, despite impressive response rates, complete responses are rare. Heat shock protein 90 (HSP90) inhibitors have been suggested as suitable therapeutic agents for NETs, as well as a potential radiosensitizers. Consequently, the aim of this study was to investigate whether the HSP90-inhibitor onalespib could reduce NET cell growth and act as a radiosensitizer when used in combination with 177Lu-DOTATATE. The NET cell lines BON, NCI-H727 and NCI-H460, were first characterized with regards to 177Lu-DOTATATE uptake and sensitivity to onalespib treatment in monolayer cell assays. The growth inhibitory effects of the monotherapies and combination treatments were then examined in three-dimensional multicellular tumor spheroids. Lastly, the molecular effects of the treatments were assessed. 177Lu-DOTATATE uptake was observed in the BON and NCI-H727 cells, while the NCI-H460 cells exhibited no detectable uptake. Accordingly, 177Lu-DOTATATE reduced the growth of BON and NCI-H727 spheroids, while no effect was observed in the NCI-H460 spheroids. Onalespib reduced cell viability and spheroid growth in all three cell lines. Furthermore, the combination of onalespib and 177Lu-DOTATATE exerted synergistic therapeutic effects on the BON and NCI-H727 spheroids. Western blot analysis of BON spheroids revealed the downregulation of epidermal growth factor receptor (EGFR) and the upregulation of γ H2A histone family member X (γH2AX) following combined treatment with onalespib and 177Lu-DOTATATE. Moreover, flow cytometric analyses revealed a two-fold increase in caspase 3/7 activity in the combination group. In conclusion, the findings of this study demonstrate that onalespib exerts antitumorigenic effects on NET cells and may thus be a feasible treatment option for NETs. Furthermore, onalespib was able to synergistically potentiate 177Lu-DOTATATE treatment in a SSTR-specific manner. The radiosensitizing mechanisms of onalespib involved the downregulation of EGFR expression and the induction of apoptosis. Consequently, the combination of onalespib and 177Lu-DOTATATE may prove to be a promising strategy with which to improve therapeutic responses in patients with NETs. Further studies investigating this strategy in vivo regarding the therapeutic effects and potential toxicities are warranted to expand these promising findings.