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Extracellular HSP90α (eHSP90α) is a promoter of tumor development and malignant progression. Patients with malignancies, including pancreatic ductal adenocarcinoma (PDAC), have generally shown 5~10-fold increases in serum/plasma eHSP90α levels. In this study, we developed a humanized antibody HH01 to target eHSP90α and evaluated its anticancer efficacy. HH01, with novel complementarity-determining regions, exhibits high binding affinity toward HSP90α. It recognizes HSP90α epitope sites 235 AEEKEDKEEE 244 and 251 ESEDKPEIED 260 , with critical amino acid residues E237, E239, D240, K241, E253, and K255. HH01 effectively suppressed eHSP90α-induced invasive and spheroid-forming activities of colorectal cancer and PDAC cell lines by blocking eHSP90α's ligation with the cell-surface receptor CD91. In mouse models, HH01 potently inhibited the tumor growth of PDAC cell grafts/xenografts promoted by endothelial-mesenchymal transition-derived cancer-associated fibroblasts while also reducing serum eHSP90α levels, reflecting its anticancer efficacy. HH01 also modulated tumor immunity by reducing M2 macrophages and reinvigorating immune T-cells. Additionally, HH01 showed low aggregation propensity, high water solubility, and a half-life time of >18 days in mouse blood. It was not cytotoxic to retinal pigmented epithelial cells and showed no obvious toxicity in mouse organs. Our data suggest that targeting eHSP90α with HH01 antibody can be a promising novel strategy for PDAC therapy.

M2-polarization and the tumoricidal to tumor-promoting transition are commonly observed with tumor-infiltrating macrophages after interplay with cancer cells or/and other stroma cells. Our previous study indicated that macrophage M2-polarization can be induced by extracellular HSP90α (eHSP90α) secreted from endothelial-to-mesenchymal transition-derived cancer-associated fibroblasts. To extend the finding, we herein validated that eHSP90α-induced M2-polarized macrophages exhibited a tumor-promoting activity and the promoted tumor tissues had significant increases in microvascular density but decreases in CD4 + T-cell level. We further investigated the signaling pathways occurring in eHSP90α-stimulated macrophages. When macrophages were exposed to eHSP90α, CD91 and toll-like receptor 4 (TLR4) functioned as the receptor/co-receptor for eHSP90α binding to recruit interleukin (IL)-1 receptor-associated kinases (IRAKs) and myeloid differentiation factor 88 (MyD88), and next elicited a canonical CD91/MyD88-IRAK1/4-IκB kinase α/β (IKKα/β)-nuclear factor-κB (NF-κB)/interferon regulatory factor 3 (IRF3) signaling pathway. Despite TLR4-MyD88 complex-associated activations of IKKα/β, NF-κB and IRF3 being well-known as involved in macrophage M1-activation, our results demonstrated that the CD91-TLR4-MyD88 complex-associated IRAK1/4-IKKα/β-NF-κB/IRF3 pathway was not only directly involved in M2-associated CD163, CD204, and IL-10 gene expressions but also required for downregulation of M1 inflammatory cytokines. Additionally, Janus kinase 2 (JAK2) and tyrosine kinase 2 (TYK2) were recruited onto MyD88 to induce the phosphorylation and activation of the transcription factor signal transducer and activator of transcription-3 (STAT-3). The JAK2/TYK2-STAT-3 signaling is known to associate with tumor promotion. In this study, the MyD88-JAK2/TYK2-STAT-3 pathway was demonstrated to contribute to eHSP90α-induced macrophage M2-polarization by regulating the expressions of M1- and M2-related genes, proangiogenic protein vascular endothelial growth factor, and phagocytosis-interfering factor Sec22b.

Octyl gallate (OG) is a common antioxidant and preservative safely used in food additive and cosmetics. In this study, OG exhibited an activity to induce apoptosis in pancreatic ductal adenocarcinoma (PDAC) cells. It induced BNIP3L level and facilitated physical associations of BNIP3L with Bcl-2 as well as Bcl-X L to set the mitochondrial Bax/Bak channels free for cytochrome c release. In addition, in vivo evaluation also showed that daily oral administration of OG was efficacious to prevent the tumor growth of PDAC cell grafts. Considering PDAC is a desmoplastic tumor consisting of many cancer-associated fibroblasts (CAFs), we further evaluated the efficacy of OG in a CAFs-involved PDAC mouse model. Endothelial-to-mesenchymal transition (EndoMT) is an important source of CAFs. The mix of EndoMT-derived CAFs with PDAC cell grafts significantly recruited myeloid-derived macrophages but prevented immune T cells. HSP90α secreted by EndoMT-derived CAFs further induced macrophage M2-polarization and more HSP90α secretion to expedite PDAC tumor growth. OG exhibited its potent efficacy against the tumor growth, M2-macrophages, and serum HSP90α level in the EndoMT-involved PDAC mouse model. CD91 and TLR4 are cell-surface receptors for extracellular HSP90α (eHSP90α). OG blocked eHSP90α-TLR4 ligation and, thus, prevented eHSP90α-induced M2-macrophages and more HSP90α secretion from macrophages and PDAC cells.

Background: Endothelial-to-mesenchymal transition (EndoMT) can provide a source of cancer-associated fibroblasts which contribute to desmoplasia of many malignancies including pancreatic ductal adenocarcinoma (PDAC). We investigated the clinical relevance of EndoMT in PDAC, and explored its underlying mechanism and therapeutic implication., Methods: Expression levels of 29 long non-coding RNAs were analyzed from the cells undergoing EndoMT, and an EndoMT index was proposed to survey its clinical associations in the PDAC patients of The Cancer Genome Atlas database. The observed clinical correlation was further confirmed by a mouse model inoculated with EndoMT cells-involved PDAC cell grafts. In vitro co-culture with EndoMT cells or treatment with the conditioned medium were performed to explore the underlying mechanism. Because secreted HSP90α was involved, anti-HSP90α antibody was evaluated for its inhibitory efficacy against the EndoMT-involved PDAC tumor., Results: A combination of low expressions of LOC340340, LOC101927256, and MNX1-AS1 was used as an EndoMT index. The clinical PDAC tissues with positive EndoMT index were significantly correlated with T4-staging and showed positive for M2-macrophage index. Our mouse model and in vitro cell-culture experiments revealed that HSP90α secreted by EndoMT cells could induce macrophage M2-polarization and more HSP90α secretion to promote PDAC tumor growth. Furthermore, anti-HSP90α antibody showed a potent therapeutic efficacy against the EndoMT and M2-macrophages-involved PDAC tumor growth., Conclusions: EndoMT cells can secrete HSP90α to harness HSP90α-overproducing M2-type macrophages to promote PDAC tumor growth, and such effect can be targeted and abolished by anti-HSP90α antibody.

Secreted levels of HSP90α and overexpression of TCF12 have been associated with the enhancement of colorectal cancer (CRC) cell migration and invasion. In this study, we observed that CRC patients with tumor TCF12 overexpression exhibited both a higher rate of metastatic occurrence and a higher average serum HSP90α level compared with patients without TCF12 overexpression. Therefore, we studied the relationship between the actions of secreted HSP90α and TCF12. Like overexpressed TCF12, secreted HSP90α or recombinant HSP90α (rHSP90α) induced fibronectin expression and repressed E-cadherin, connexin-26, connexin-43, and gap junction levels in CRC cells. Consistently, rHSP90α stimulated invasive outgrowths of CRC cells from spherical structures during three-dimensional culture. rHSP90α also induced TCF12 expression in CRC cells. Its effects on CRC cell epithelial-mesenchymal transition, migration, and invasion were drastically prevented when TCF12 was knocked down. This suggests that TCF12 expression is required for secreted HSP90α to enhance CRC cell spreading. Through the cellular receptor CD91, rHSP90α facilitated the complex formation of CD91 with IκB kinases (IKKs) α and β and increased the levels of phosphorylated (active) IKKα/β and NF-κB. Use of an IKKα/β inhibitor or ectopic overexpression of dominant-negative IκBα efficiently repressed rHSP90α-induced TCF12 expression. Moreover, κB motifs were recognized in the gene sequence of the TCF12 promoter, and a physical association between NF-κB and the TCF12 promoter was detected in rHSP90α-treated CRC cells. Together, these results suggest that the CD91/IKK/NF-κB signaling cascade is involved in secreted HSP90α-induced TCF12 expression, leading to E-cadherin down-regulation and enhanced CRC cell migration/invasion.

Purpose: We studied whether HSP90α was associated with the special carbohydrate structures IMH-2 epitopes, and investigated its mRNA expression and clinical relevance in colorectal cancer (CRC) patients., Methods: The lysates and the culture media of colon cancer HCT-8 cells were immunoprecipitated with IMH-2 antibody, and the immunoprecipitates were subsequently analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or by immunoblotting with anti-HSP90α antibody. In vitro wound-healing assay was done to evaluate the role of IMH-2 epitope-associated HSP90α in HCT-8 cell migration. Real-time RT-PCR was performed to detect the levels of HSP90α mRNA expression in paired tumor and non-tumor tissues of 56 CRC patients. The correlation of tumor HSP90α mRNA overexpression with CRC metastasis and poor survival outcome was determined by statistical analyses., Results: HSP90α was first identified as an IMH-2 epitope-associated protein by immunoprecipiation, mass spectrometry, and immunoblotting analysis. IMH-2 epitopes were detected in both cellular and secreted HSP90α. HCT-8 cell migration induced by serum starvation-conditioned medium was blocked by anti-HSP90α antibody or the HSP90α inhibitor geldanamycin (GA) as efficient as by IMH-2 antibody, suggesting that IMH-2-associated HSP90α was involved in serum starvation-induced CRC cell migration. On the other hand, HSP90α mRNA expression was induced in HCT-8 cells after serum starvation. Clinically, 15 (26.8%) of 56 CRC patients exhibited tumor HSP90α mRNA overexpression and had higher rates of metastatic occurrence (P = 0.003) and poor prognosis (P = 0.028)., Conclusions: HSP90α was an IMH-2 epitope-associated protein. Tumor HSP90α overexpression was correlated with the metastasis and poor prognosis of CRC patients.

HCT-8 colon cancer cells secreted heat shock protein 90alpha (HSP90alpha) and had increased invasiveness upon serum starvation. The concentrated conditioned medium of serum-starved HCT-8 cells was able to stimulate the migration and invasion of non-serum-starved cells, which could be prevented by treatment with an anti-HSP90alpha antibody. Recombinant HSP90alpha (rHSP90alpha) also enhanced HCT-8 cell migration and invasion, suggesting a stimulatory role of secreted HSP90alpha in cancer malignancy. HSP90alpha binding to CD91alpha and Neu was evidenced by the proximity ligation assay, and rHSP90alpha-induced HCT-8 cell invasion could be suppressed by the addition of anti-CD91alpha or anti-Neu antibodies. Via CD91alpha and Neu, rHSP90alpha selectively induced integrin alpha(V) expression, and knockdown of integrin alpha(V) efficiently blocked rHSP90alpha-induced HCT-8 cell invasion. rHSP90alpha induced the activities of ERK, PI3K/Akt, and NF-kappaB p65, but only NF-kappaB activation was involved in HSP90alpha-induced integrin alpha(V) expression. Additionally, we investigated the serum levels of HSP90alpha and the expression status of tumor integrin alpha(V) mRNA in colorectal cancer patients. Serum HSP90alpha levels of colorectal cancer patients were significantly higher than those of normal volunteers (p < 0.001). Patients with higher serum HSP90alpha levels significantly exhibited elevated levels of integrin alpha(V) mRNA in tumor tissues as compared with adjacent non-tumor tissues (p < 0.001). Furthermore, tumor integrin alpha(V) overexpression was significantly correlated with TNM (Tumor, Node, Metastasis) staging (p = 0.001).