Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non-small cell lung carcinoma (NSCLC); an implication for NSCLC treatment.

Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.

HSP70i is a critical component of the immune response leading to vitiligo.

HSP70i and other stress proteins have been used in anti-tumor vaccines. This begs the question whether HSP70i plays a unique role in immune activation. We vaccinated inducible HSP70i (Hsp70-1) knockout mice and wild-type animals with optimized TRP-1, a highly immunogenic melanosomal target molecule. We were unable to induce robust and lasting depigmentation in the Hsp70-1 knockout mice, and in vivo cytolytic assays revealed a lack of cytotoxic T-lymphocyte activity. Absence of T-cell infiltration to the skin and maintenance of hair follicle melanocytes were observed. By contrast, depigmentation proceeded without interruption in mice lacking a tissue-specific constitutive isoform of HSP70 (Hsp70-2) vaccinated with TRP-2. Next, we demonstrated that HSP70i was necessary and sufficient to accelerate depigmentation in vitiligo-prone Pmel-1 mice, accompanied by lasting phenotypic changes in dendritic cell subpopulations. In summary, these studies assign a unique function to HSP70i in vitiligo and identify HSP70i as a targetable entity for treatment.

HSP72 depletion suppresses gammaH2AX activation by genotoxic stresses via p53/p21 signaling.

Knockout of heat shock protein Hsp72 was shown to promote chromosomal instability and increase radiation sensitivity of mouse fibroblasts. Here, we report that downregulation of Hsp72 in human tumor cells leads to suppression of a specific branch of the DNA damage response (DDR) that facilitates DNA repair following genotoxic insults, that is, reduced accumulation of the phosphorylated form of histone H2AX (gammaH2AX). This inhibition was due to decreased expression of H2AX as well as higher rate of gammaH2AX dephosphorylation. Formation of gammaH2AX and MDC1 radiation-induced foci was impaired in Hsp72-depleted cells, which in turn enhanced DNA damage, resulting in sensitization of cells to gamma-radiation and doxorubicin. These effects of Hsp72 knockdown were dependent on activation of the p53/p21-signaling pathway. Overall, permanent activation of the p53/p21 signaling in Hsp72-depleted cells specifically impaired the gammaH2AX pathway of the DDR, enhanced DNA damage following genotoxic insults, and led to further stimulation of the p53/p21 pathway, thus creating a positive feedback loop. The resulting strong induction of p21 precipitated senescence following exposure to DNA-damaging agents, thus accounting for higher sensitivity of cells to genotoxic stresses.

Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease.

Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.

Activation of histone deacetylase 2 by inducible heat shock protein 70 in cardiac hypertrophy.

Diverse cardiac diseases induce cardiac hypertrophy, which leads to dilatation and heart failure. We previously reported that hypertrophy can be blocked by class I histone deacetylase (HDAC) inhibitor, which prompted us to investigate the regulatory mechanism of class I HDACs. Cardiac hypertrophy was introduced by aortic banding, by infusion of isoproterenol or angiotensin II, or by swimming. Hypertrophic stimuli transiently elevated the activity of histone deacetylase-2 (Hdac2), a class I HDAC. In cardiomyocytes, forced expression of Hdac2 simulated hypertrophy in an Akt-dependent manner, whereas enzymatically inert Hdac2 H141A failed to do so. Hypertrophic stimuli induced the expression of heat shock protein (Hsp)70. The induced Hsp70 physically associated with and activated Hdac2. Hsp70 overexpression produced a hypertrophic phenotype, which was blocked either by siHdac2 or by a dominant negative Hsp70DeltaABD. In Hsp70.1(-/-) mice, cardiac hypertrophy and Hdac2 activation were significantly blunted. Heat shock either to cardiomyocytes or to mice activated Hdac2 and induced hypertrophy. However, heat shock-induced Hdac2 activation was blunted in the cardiomyocytes isolated from Hsp70.1(-/-) mice. These results suggest that the induction of Hsp70 in response to diverse hypertrophic stresses and the ensuing activation of HDAC2 trigger cardiac hypertrophy, emphasizing HSP70/HDAC2 as a novel mechanism regulating hypertrophy.

Triggering senescence programs suppresses Chk1 kinase and sensitizes cells to genotoxic stresses.

Depletion of the major heat shock protein Hsp72 leads to activation of the senescence program in a variety of tumor cell lines via both p53-dependent and p53-independent pathways. Here, we found that the Hsp72-depleted cells show defect in phosphorylation and activation of the protein kinase Chk1 by genotoxic stresses, such as UVC irradiation or camptothecin. Under these conditions, phosphorylation of Rad17 was also suppressed, whereas phosphorylation of p53 at Ser(15) was not affected, indicating a specific defect in phosphorylation of a subset of the ATR kinase substrates. Similarly, suppression of Chk1 activation was seen when senescence signaling was triggered by direct stimulation of p53, depletion of Cdc2, or overexpression of the cell cycle inhibitors p21 or p16. Thus, defect in Chk1 activation was not a consequence of the chaperone imbalance, but rather a downstream effect of activation of the senescence signaling. Inhibition of Chk1 was associated with inefficient inter-S phase checkpoint, as Hsp72 depleted cells failed to halt cell cycle progression upon UVC irradiation. Accordingly, sensitivity of cells to genotoxic stimuli after Hsp72 depletion was significantly enhanced. Thus, activation of the senescence signaling causes a defect in the DNA damage response manifested in increased sensitivity to genotoxic stresses.