HSP70s in Breast Cancer: Promoters of Tumorigenesis and Potential Targets/Tools for Therapy

The high frequency of breast cancer worldwide and the high mortality among women with this malignancy are a serious challenge for modern medicine. A deeper understanding of the mechanisms of carcinogenesis and emergence of metastatic, therapy-resistant breast cancers would help development of novel approaches to better treatment of this disease. The review is dedicated to the role of members of the heat shock protein 70 subfamily (HSP70s or HSPA), mainly inducible HSP70, glucose-regulated protein 78 (GRP78 or HSPA5) and GRP75 (HSPA9 or mortalin), in the development and pathogenesis of breast cancer. Various HSP70-mediated cellular mechanisms and pathways which contribute to the oncogenic transformation of mammary gland epithelium are reviewed, as well as their role in the development of human breast carcinomas with invasive, metastatic traits along with the resistance to host immunity and conventional therapeutics. Additionally, intracellular and cell surface HSP70s are considered as potential targets for therapy or sensitization of breast cancer. We also discuss a clinical implication of Hsp70s and approaches to targeting breast cancer with gene vectors or nanoparticles downregulating HSP70s, natural or synthetic (small molecule) inhibitors of HSP70s, HSP70-binding antibodies, HSP70-derived peptides, and HSP70-based vaccines

Induction of Hsp70 in tumor cells treated with inhibitors of the Hsp90 activity: A predictive marker and promising target for radiosensitization.

We studied a role of the inducible heat shock protein 70 (Hsp70) in cellular response to radiosensitizing treatments with inhibitors of the heat shock protein 90 (Hsp90) chaperone activity. Cell lines derived from solid tumors of different origin were treated with the Hsp90 inhibitors (17AAG, geldanamycin, radicicol, NVP-AUY922) or/and γ-photon radiation. For comparison, human cells of the non-cancerous origin were subjected to the same treatments. We found that the Hsp90 inhibitors yielded considerable radiosensitization only when they cause early and pronounced Hsp70 induction; moreover, a magnitude of radiosensitization was positively correlated with the level of Hsp70 induction. The quantification of Hsp70 levels in Hsp90 inhibitor-treated normal and cancer cells enabled to predict which of them will be susceptible to any Hsp90-inhibiting radiosensitizer as well as what concentrations of the inhibitors ensure the preferential cytotoxicity in the irradiated tumors without aggravating radiation damage to adjacent normal tissues. Importantly, the Hsp70 induction in the Hsp90 inhibitor-treated cancer cells appears to be their protective response that alleviates the tumor-sensitizing effects of the Hsp90 inactivation. Combination of the Hsp70-inducing inhibitors of Hsp90 with known inhibitors of the Hsp induction such as quercetin, triptolide, KNK437, NZ28 prevented up-regulation of Hsp70 in the cancer cells thereby increasing their post-radiation apoptotic/necrotic death and decreasing their post-radiation viability/clonogenicity. Similarly, co-treatment with the two inhibitors conferred the enhanced radiosensitization of proliferating rather than quiescent human vascular endothelial cells which may be used for suppressing the tumor-stimulated angiogenesis. Thus, the easily immunodetectable Hsp70 induction can be a useful marker for predicting effects of Hsp90-inhibiting radiosensitizers on tumors and normal tissues exposed to ionizing radiation. Moreover, targeting the Hsp70 induction in Hsp90 inhibitor-treated cancer cells and tumor vasculature cells may beneficially enhance the radiosensitizing effect

Effects of the Hsp90 activity inhibition with 17AAG and suppression of the Hsp70 induction with quercetin (Querc) on post-radiation apoptosis and necrosis in MCF-7 breast cancer cells.

<p>Effects of the Hsp90 activity inhibition with 17AAG and suppression of the Hsp70 induction with quercetin (Querc) on post-radiation apoptosis and necrosis in MCF-7 breast cancer cells.</p

Texas-Red-immunofluorescence patterns of inducible Hsp70 in fixed/permeabilized cells of different cell lines.

<p>The cells were immunostained as untreated (control) samples or after 20 h incubation with 100 nM 17AAG without quercetin or in the presence of 40 μM quercetin, or 20 h after hyperthermia (43°C, 60 min); bar = 10 μm. It is clearly seen that the marked Hsp70 induction (the brightly stained cytoplasm) takes place in all cell samples exposed to hyperthermia as well as in HeLa cells and MCF-7 cells incubated with 17AAG, whereas 17AAG-treated HBL-100 cells and 293 cells are not stained. Very similar results were obtained with 50–200 nM geldanamycin or 30–100 nM radicicol, or 20–100 nM NVP-AUY922 instead of 17AAG and with 3–10 nM triptolide or 100–200 μM KNK437, or 5–20 μM NZ28 instead of quercetin (not shown). Similar variability in the expression of inducible Hsp70 was found in other cell cultures (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173640#pone.0173640.t001" target="_blank">Table 1</a>).</p

The data of Western blotting and MTT assay demonstrating the enhanced radiosensitization of 17AAG-treated HeLa cells by preventing the Hsp70 induction with triptolide or quercetin.

<p>The presented blots (<b>A</b>) demonstrate that both inhibitors of the Hsp induction completely abrogated up-regulation of inducible Hsp70 in response to the 17AAG treatment. (The values of Hsp70/Actin band ratio are presented along the lower sides of blots and reflect the relative amount of Hsp70 in cell samples. Of note, such co-treatments with the Hsp70 induction inhibitors did not decrease the basal level of constitutively expressed Hsp70 in target cells). The presented curves (<b>B</b>) show that in contrast to the action of 17AAG alone, the two-inhibitor combinations prevented the post-radiation recovery of proliferative activity in the drug-treated cells. Very similar results were obtained with 50–200 nM geldanamycin or 30–100 nM radicicol, or 50–200 nM NVP-AUY922 instead of 17AAG and with 100–200 μM KNK437, or 5–20 μM NZ28 instead of quercetin and triptolide (not shown). MTT assay also revealed the enhanced radiosensitization of MCF-7, KTC-1, PC-3, Myc-CaP and HT 1080 cancer cells pretreated with combination of the Hsp90 activity inhibitors and inhibitors of the Hsp70 induction (not shown). The presented data express mean ± SEM of 5 independent experiments. *—significant difference from the respective unmarked values, p<0.05; **—significant difference from the respective unmarked or marked with * values, p<0.05.</p

Western blots showing the diversity in dose-dependent effects of 17AAG on the Hsp70 induction in cancer MCF-7 cells and non-cancer 293 cells.

<p>The cells were lysed at different time points (indicated in hours along the upper sides of blots) of incubation with graded concentrations 17AAG and then analyzed with antibodies to inducible Hsp70 and β-Actin (load control). The values of Hsp70/Actin band ratio are presented along the lower sides of blots and reflect the relative amount of Hsp70 in cell samples. As it is seen, Hsp70 is induced in MCF-7 cells by much lower concentrations of 17AAG as compared to 293 cells. The similar difference was observed with other Hsp90 inhibitors in other cell cultures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173640#pone.0173640.t001" target="_blank">Table 1</a>).</p

Targeting the Hsp70 induction in Hsp90 inhibitor-treated cancer cells enhances their apoptotic death following radiation exposure.

<p>MCF-7 breast cancer cells were either untreated (control) or exposed to γ-photons (6 Gy) without any drug pretreatment or after 24 h incubation with 50 nM 17AAG alone or in combination with 40 μM quercetin (<b>Q</b>). After 48 h, the cells were stained with FITC-annexin V/propidium iodide (PI) and analyzed by flow cytometry. The presented distribution of stained cell subpopulations demonstrates the considerable enhancement of post-radiation apoptosis (FITC-annexin V-positive, PI-negative cells) and secondary necrosis (PI-positive cells) in samples where the 17AAG-induced up-regulation of Hsp70 was fully blocked by quercetin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173640#pone.0173640.t003" target="_blank">Table 3</a>). Analogous effects were also observed on HeLa, PC-3 and Myc-CaP cancer cells and actively proliferating vascular endothelial cells (not shown).</p

Various cell cultures can exhibit the different susceptibility of their Hsp90 chaperone machine and their HSF1-mediated Hsp70 induction to inhibitors of the Hsp90 activity.

<p>Here it is seen that much lower concentrations of 17AAG are required to repress the Hsp90 chaperone function-dependent refolding of luciferase (<b>A</b>) and stimulate the HSF1 phosphorylation (<b>B</b>) and the Hsp70 induction (<b>C</b>) in MCF-7 breast cancer cells as compared with non-cancerous 293 cells. Numbers under the blots represent the expression of phosphorylated HSF1 (pHSF1) or inducible Hsp70 relative to β-actin. Both cell cultures were treated with 17AAG for 20 h before analyses.</p