The Chaperone System in Salivary Glands: Hsp90 Prospects for Differential Diagnosis and Treatment of Malignant Tumors

Abstract

Salivary gland tumors represent a serious medical problem and new tools for differential diagnosis and patient monitoring are needed. Here, we present data and discuss the potential of molecular chaperones as biomarkers and therapeutic targets, focusing on Hsp10 and Hsp90. The salivary glands are key physiological elements but, unfortunately, the information and the means available for the management of their pathologies, including cancer, are scarce. Progress in the study of carcinogenesis has occurred on various fronts lately, one of which has been the identification of the chaperone system (CS) as a physiological system with presence in all cells and tissues (including the salivary glands) that plays a role in tumor-cell biology. The chief components of the CS are the molecular chaperones, some of which belong to families of evolutionarily related molecules named heat shock protein (Hsp). We are quantifying and mapping these molecular chaperones in salivary glands to determine their possible role in the carcinogenetic mechanisms in these glands and to assess their potential as diagnostic biomarkers and therapeutic targets. Here, we report recent findings on Hsp10 and Hsp90 and show that the quantitative and topographic patterns of tissue Hsp90 are distinctive of malignant tumors and differentiate benign from malignant lesions. The Hsp90 results show a correlation between quantity of chaperone and tumor progression, which in turn calls for negative chaperonotherapy, namely, elimination/inhibition of the chaperone to stop the tumor. We found that in vitro, the Hsp90 inhibitor Ganetespib is cytotoxic for the salivary gland UM-HACC-2A cell line. The drug, by interfering with the pro-survival NF-κB pathway, hampers cellular proliferation and migration, and favors apoptosis, and can, therefore, be considered a suitable candidate for future experimentation to develop a treatment for salivary gland tumors.

Keywords: Ganetespib; Hsp90; Hsp90 biomarker; Hsp90 pathogenic; chaperone system; differential diagnosis; negative chaperonotherapy; salivary gland tumors.

Figure 1

Immunohistochemical determinations of Hsp10 and Hsp90 in adult human SMG and PG. H&E (hematoxylin-eosin staining) of normal (Ctrl) SMG and PG (A). Immunostaining of normal SMG and PG shows that Hsp10 (B,a) and Hsp90 (C,b) are present in the cytoplasmic portion of the ducts and in the acinar cells (arrowheads). H&E of sialadenitis (Sial) (D). Hsp10 (E,a) and Hsp90 (F,b) are present in the cytoplasm of the ducts and acini (arrowheads). H&E of Warthin’s tumor (WT) (G). Hsp10 (H,c) and Hsp90 (I,d) are present in the epithelium of the tumoral tissue (arrowheads). H&E of pleomorphic adenoma (PA) (J). Hsp10 (K,e) and Hsp90 (L,f) are present in the epithelium of the tumoral tissue (arrowheads). H&E of carcinoma ex-pleomorphic adenoma (EX PA) (M). Hsp10 (N,g) and Hsp90 (O,h) are strongly positive in the cytoplasm of the neoplastic cells (arrowheads). H&E of mucoepidermoid carcinoma (MUC) (P). Hsp10 (Q,i) and Hsp90 (R,j) are present in the cytoplasm of the epithelium of the neoplastic cells (arrowheads). H&E of adenoid cystic carcinoma (ACC) (S). Hsp10 (T,k) and Hsp90 (U,l) occur in the cytoplasm of the neoplastic cells (arrowheads). * Significantly different (p ≤ 0.005), ** (p ≤ 0.05), *** (p ≤ 0.001), **** (p ≤ 0.0005). Bar 20 μm.

Figure 2

Left. Confocal microscopy analysis for Hsp90 in human adult SMG and PG. Hsp90 immunostaining in Normal (A), WT (B), PA (C), EX-PA (D), MUC (E), and ACC (F). Bar 25 μm. Right. Histogram. The staining intensity for Hsp90 (bars) in Normal (G), WT (G), PA (H), EX-PA (I), MUC (J), ACC (K) was expressed as the mean pixel intensity (PI) normalized to the CSA (cross-sectional area), using the software Leica Application Suite Advanced Fluorescences software. Data are presented as the mean ± SD. * Significantly different (p ≤ 0.005), ** (p ≤ 0.05). Abbreviations: Normal, normal submandibular and parotid gland; WT, Warthin’s tumor; PA, pleomorphic adenoma; EX-PA, carcinoma ex-pleomorphic adenoma; MUC, mucoepidermoid carcinoma; ACC, adenoid cystic carcinoma.

Figure 3

Ganetespib induces apoptosis in UM-HACC-2A cells. (A) Histogram representing the percentage of cell viability upon treatment with ascending concentrations of Ganetespib (0, 15, 30, 60, 120, and 150 nM) for 24 h. (B) Representative figures of DAPI nuclear staining of Ganetespib-treated and non-treated UM-HACC-2A cells. Apoptosis was assessed by nuclear fragmentation (indicated by white arrows), chromatic condensation (indicated by red arrows), and apoptotic bodies (indicated by green arrows). (C) Histogram representing the percentage of apoptotic cells reported as the apoptotic index. (D) Representative figures of TUNEL cell death detection assay of Ganetespib-treated and non-treated UM-HACC-2A cells. (E) Histogram representing the percentage of apoptotic cells reported as the apoptotic index. Abbreviations: PC, positive control; NC, negative control; NT, non-treated; T, treated. ** Significantly different (p ≤ 0.01), *** (p ≤ 0.0001). Bar 20 μm.

Figure 4

Ganetespib reduces UM-HACC-2A cells proliferation and migration. (A) Panel representing non-treated vs. treated cells after 24 h Ganetespib treatment. (B) Histogram representing cell counts after 24 h of Ganetespib treatment. (C) Panel representing wound-healing assay showing cellular migration at 0 and 24 h after Ganetespib treatment. (D) Histogram representing the gap filling scores of cells at 0 and 24 h after Ganetespib treatment. ** Significantly different (p ≤ 0.005), *** (p ≤ 0.001).

Figure 5

Protein alterations in Ganetespib-treated UM-HACC-2A cells. (A) Representative Western blot of protein expression in NT vs. T UM-HACC-2A cells. Histogram representing Hsp90 (B), Hsp70 (C), p-akt/t-akt (D), NF-κB (E), c-cas3/p-cas3 (F), and VEGF (G) protein levels in Ganetespib-treated UM-HACC-2A cells vs. non-treated cells. Abbreviations: NT, non-treated; T, treated; p-ak13hosphorpho Akt; t-akt, total Akt; c-cas3, cleaved caspase 3; p-cas3, pro-caspase 3. * Significantly different (p ≤ 0.05).