Nanofitins targeting heat shock protein 110: An innovative immunotherapeutic modality in cancer.

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds.

Selecting the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy.

Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.

Nuclear heat shock protein 110 expression is associated with poor prognosis and hyperthermo-chemotherapy resistance in gastric cancer patients with peritoneal metastasis.

AIM: To investigate the significance of heat shock protein 110 (HSP110) in gastric cancer (GC) patients with peritoneal metastasis undergoing hyperthermo-chemotherapy. METHODS: Primary GC patients (n = 14) with peritoneal metastasis or positive peritoneal lavage cytology who underwent distal or total gastrectomy between April 2000 and December 2011 were enrolled in this study. The patients underwent postoperative intraperitoneal hyperthermo-chemotherapy using a Thermotron RF-8 heating device two weeks after surgery. We analyzed nuclear HSP110 expression in surgically resected tumors using immunohistochemistry. Additionally, the effect of HSP110 suppression on hyptherthermo-chemosensitivity was assessed in vitro in the MKN45 GC cell line using the HSP inhibitor KNK437. RESULTS: HSP110 immnohistochemical staining in 14 GC patients showed that five (35.7%) samples belonged to the low expression group, and nine (64.3%) samples belonged to the high expression group. Progression-free survival was significantly shorter in the HSP110 high-expression group than in the low-expression group (P = 0.0313). However, no significant relationships were identified between HSP110 expression and the clinicopathological characteristics of patients. Furthermore, high HSP110 expression was not an independent prognostic factor in GC patients with peritoneal metastasis (P = 0.0625). HSP110 expression in MKN45 cells was suppressed by KNK437 at the hyperthermic temperature of 43 °C in vitro. Comparison of MKN45 cell proliferation in the presence and absence of KNK437 at 43 °C, revealed that proliferation was significantly decreased when HSP110 was inhibited by KNK437. Additionally, HSP110 suppression via HSP inhibitor treatment increased cellular sensitivity to hyperthermo-chemotherapy in vitro. CONCLUSION: The expression of nuclear HSP110 in GC patients might be a new marker of chemosensitivity and a therapeutic target for patients who are tolerant to existing hyperthermo-chemotherapies.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo.

We previously reported that heat shock protein 105 (HSP105), identified by serological analysis of a recombinant cDNA expression library (SEREX) using serum from a pancreatic cancer patient, was overexpressed in various human tumors and in the testis of adult men by immunohistochemical analysis. In the present study, to elucidate the biological function of the HSP105 protein in cancer cells, we first established NIH3T3 cells overexpressing murine HSP105 (NIH3T3-HSP105). The NIH3T3-HSP105 cells acquired resistance to apoptosis induced by heat shock or doxorubicin. The small interfering RNA (siRNA)-mediated suppression of HSP105 protein expression induced apoptosis in human cancer cells but not in fibroblasts. By a combination of siRNA introduction and doxorubicin or heat shock treatment, apoptosis was induced synergistically in a human colon cancer cell line, HCT116. In vivo, siRNA inoculation into the human gastric cancer cell line KATO-3 established in the flank of an NOD SCID mouse suppressed the tumor growth. This siRNA-induced apoptosis was mediated through caspases, but not the p53 tumor suppressor protein, even though the HSP105 protein was bound to wild-type p53 protein in HCT116 cells. These findings suggest that the constitutive overexpression of HSP105 in cancer cells is involved in malignant transformation by protecting tumor cells from apoptosis. HSP105 may thus be a novel target molecule for cancer therapy and a treatment regimen using synthetic siRNA to suppress the expression of HSP105 protein may provide a new strategy for cancer therapy.