HSP40: Drug Discovery

Due to the large number of Dna/Hsp40 isoforms and splice variants that often show opposing effects, DNAJs/HSP40s appear to be less “druggable”. As a consequence, attempts to target  DNAJs/HSP40s are infrequent. With respect to cancer, the downregulation of certain DNAJs/HSP40s have been shown to correlate with a highly aggressive and invasive phenotype (see Disease Relevance). As already mentioned, the cancer-testis antigen DnaJB8/Dj6 contributes to the cancer stem-like cell (CSC) phenotype in renal cell carcinoma (RCC) 361. CSCs are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. By using an immunological vaccination approach, Nishizawa and colleagues could demonstrate a much stronger anti-tumor effect in mice immunized with DnaJB8/Dj6 compared to immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC 361. These findings imply a crucial role of DnaJB8/Dj6 in tumor initiation and CSC maintenance rendering it a candidate for CSC-targeting immunotherapy in RCC 361. The same group identified a preferential expression of DnaJB8/Dj6 in CSCs/cancer-initiating cells (CICs) derived from colorectal cancer (CRC) cells rather than in non-CSCs/CICs 362. CSCs/CICs are thought to play a pivotal role in tumor maintenance, recurrence and distant metastasis. In this study, overexpression of DnsJB8/Dj6 enhanced the expression of stem cell markers and tumorigenicity highlighting the importance of  DnaJB8 /Dj6 in CRC CSCs/CICs 362. A DnaJB8/Dj6-derived antigenic peptide has been shown to induce a DnaJB8/Dj6-specific cytotoxic T lymphocyte (CTL) response. Remarkably, adoptive transfer of CTLs into CRC CSCs/CICs showed an anti-tumor effect in vivo rendering DnaJB8/Dj6 as being a novel target of CRC CSCs/CICs-targeting immunotherapy 362.

Several investigations in the past years approved DnaJ as being a useful candidate vaccine protein. It has been shown previously that intraperitoneal or intranasal immunization with recombinant DnaJ induces a striking protective immune response and protects mice against focal and lethal infections with different serotypes of S. pneumoniae 379, 380. The mucosal immunization with DnaJ antigen yielded the generation of both, systemic and mucosal antibodies against DnaJ and stimulated the release of high levels of IL-10, IFN-γ and IL-17A 379. DnaJ plays an important role in the pathogenesis of pneumococcal infections 11, and the antibody to DnaJ could inhibit S. pneumoniae adhesion to epithelial lung carcinoma cells 379. It is worth mentioning that mucosal immunization with recombinant fusion protein DnaJ-ΔA146Ply has been identified to boost IL-17A-mediated cross-protective immunity against S. pneumoniae infection in mice 381.

Polyglutamine (polyQ) diseases such as Huntington’s disease (HD) are neurodegenerative diseases caused by proteins with an expanded polyQ stretch that misfold, aggregate, and subsequently accumulate as inclusion bodies within neurons 382. Several strategies against downstream targets of the pathogenic cascade have been conducted in recent years. Nevertheless, misfolded and aggregated polyQ proteins are ideal therapeutic targets because their formation represents the most initial pathogenic events whose inhibition is thought to block a broad range of downstream pathogenic events 383, 384. In an attempt to establish a therapy for polyQ diseases, Popiel and collaborators employed a viral vector to deliver a DNAJ/HSP40 transgene into the brain and investigated its therapeutic effect on polyQ disease (HD) model mice. They found that injection of Hsp40 into the striatum dramatically suppressed inclusion body formation in HD model mice, ameliorated the motor impairment and extended the lifespan of the animals 385. Furthermore, Hsp40 inhibited secretion of polyQ proteins from cultured cells implying the suppression of the recently suggested cell-cell transmission of the polyQ protein. These data clearly reveal the therapeutic effectiveness of DNAJ/HSP40 gene therapy on the neurological phenotypes of polyQ disease mice 385.

In recent years a wealth of evidence has been collected to demonstrate that HSPs are co-localized in protein aggregates in Alzheimer’s disease (AD), Parkinson’s disease (PD), polyQ disease, prion disease, and other neurodegenerative disorders (for a review see Paul and Mahanta, 2014 386). Experimental data have already established that selective upregulation of HSPs such as DnaJ/Hsp40 and Hsp70 prevents disease progression in several cellular and animal models. Various HSP modulators like geldanamycin, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-AAG), radicicol (bortezomib,  velcade), and celastrol have recently been shown to augment the expression of DnaJ/Hsp40 and Hsp70 which in turn triggers the solubilization of diseased protein aggregates 386. For instance, the HSF-1 stimulator celastrol increased transcription of the amyloid precursor transthyretin (TTR) in parallel with that of DNAJ/HSP40, HSP70, and HSP90 in human neuroblastoma cells 387. Increased production of TTR has been suggested in neurodegenerative diseases, particularly AD, by different groups 388, 389, 390, 391. The function of TTR as a neuronal stress protein has been confirmed in an animal model of AD suggesting one possible mechanism that can account for the increased production of TTR as a potential protective molecule during the course of AD 387. These findings further support the idea that TTR can interfere with the aggregation and blocks the toxicity of Aβ and its oligomers in vivo. Recent studies by the Durham group demonstrated that three different Hsp90 inhibitors (geldanamycin, 17-AAG, and radicicol) induced expression of Hsp70 and its co-chaperone Hsp40 in cultured motor neurons, presumably through activation of HSF-1 thereby conferring neuroprotection 392.

Figure 6.  (click image for larger version)HSP40 Drug Discovery - Quercetin, KNK437, butyl 3-[2-(2,4-dichlorophenoxy)acetamido]benzoate 
Pharmacological approaches by the group of Manoj Kumar Bhat yielded an upregulation of Hsp40 and HspB1/Hsp27 in hepatoma cells exposed to 5-fluorouracil (5-FU) or carboplatin allowing their survival 393. In this regard, exposure of cells to the chemicals ibuprofen, indomethacin, ZnSO4, and 8-hydroxy-quinoline induced expression of Hsp40/DnaJB1 with concomitant translocation to the nucleus 191. Moreover, the ER stress gene DNAJC10/ERDJ5 was found to be induced in neuroectodermal tumor cells by the retinoid analog fenretinide 192, which has an increasingly important profile as a cancer preventive and chemotherapeutic drug 193. Downregulation of Hsp40 and HspB1/Hsp27 by the polyphenolic  bioflavanoid  compound quercetin (3,3′,4′,5,7-pentahydroxyflavon; Figure 6) induced cytotoxicity in hepatoma cells and endowed a better outcome to 5-FU or carboplatin treatment. The protective action of both chaperones has been confirmed by siRNA-mediated knockdown of Hsp40 and HspB1/Hsp27 393. It is well documented that quercetin suppresses the expression of HSPs in cells by depleting cellular stores of HSF-1 394, 395. Curcumin, a non-flavonoid polyphenol from turmeric Curcuma longa with anti-tumor capacity, has recently been identified to upregulate DnaJB4/Hlj1 expression in human lung adenocarcinoma cells through activation of the JNK/JunD signaling pathway thereby blocking lung cancer cell invasion and metastasis by modulating E-cadherin expression 260. Most recently, curcumin inhibited the proliferation and invasion of NSCLC cells through metastasis-associated protein 1 (MTA-1)-mediated inactivation of the Wnt/β-catenin pathway 396. MTA-1 overexpression has been detected in a wide variety of aggressive tumors and plays a crucial role in cell invasion and metastasis 397. The data by Lu et al. provide evidence for the critical cooperative role of Wnt/β-catenin signaling in promoting lung tumorigenesis 396. These novel mechanisms support the application of curcumin as a promising candidate to supplement standard therapeutic concepts in NSCLC.

Prenylation is a characteristic post-translational modification of type 1 DNAJs/HSP40s. It has recently been shown that in vivo simvastatin treatment resulted in inhibition of geranylgeranylation in murine mononuclear cells (MNCs) and MNCs from a subset of patients with acute myeloid leukemia (AML) 398. In addition, two out of seven patients displayed an increased in vitro chemosensitivity of their AML cells upon simvastatin treatment. Form these findings one can conclude that simvastatin inhibits the prenylation of DNAJs/HSP40s leading to chemosensitization in a subset of AML patients. Addition of an isoprenyl (farnesyl or geranylgeranyl) lipid group to certain cysteine residues in proteins like Ras and HSPs confers on them a membrane address required for correct function. Chronic activation of Ras drives tumorigenesis of a broad panel of cancers including glioblastoma multiforme (reviewed by Grabocka et al., 2014 399). In this regard, farnesyltransferase inhibitors (FTI) were introduced already 20 years ago in preclinical and clinical trials to target aberrant Ras in order to block signaling of tumor cell proliferation, invasion, and angiogenesis 400. Although clinical trials of R115777 (Tipifarnib™, Zarnestra), a nonpeptidomimetic FTI, revealed promising effects in hematologic malignancies 401, 402, phase II and III studies in patients with lung cancer, colorectal, or pancreatic cancer did not convincingly show single-agent efficacy 403, 404, 405, 406. The studies by Wang and co-workers indicated that R115777 can radiosensitize glioma cell lines via the p53/p21 pathway involving the type 1 DNAJ/HSP40 family member DnaJA1/Hdj2 371. The obvious contribution of DnaJA1/Hdj2 was strengthened by the finding that cellular radiosensitivity was enhanced by genetic inhibition of DnajA1/Hdj2, whereas overexpression conferred radioresistance. Of note, irradiation of cells induced the translocation of DnaJA1/Hdj2 from the cytoplasm to the nucleus, and this migration could be prevented by prior FTI treatment 371. Decreased farnesylation of DnaJA1/Hdj2 has also been reported in pancreatic adenocarcinoma cells after exposure to R115777 407. In fact, treatment with R115777 resulted in partial inhibition of farnesyltransferase activity in mononuclear cells, but it did not exhibit single-agent anti-tumor activity in patients with previously untreated metastatic pancreatic cancer. These disillusioning results may at least help to explain the variability in responses to FTIs.

DnaJB4/Hlj1 has emerged as a novel tumor suppressor and a potential druggable target for NSCLC. By using a promoter-containing enhancer region as the DnaJB4/Hlj1-targeting drug-screening platform, Lai et al. identified numerous herbal compounds from a Chinese herbal bank with the capacity to enhance DNAJB4/HLJ1 promoter activity and suppress tumor growth and invasion of NSCLC 408. Amongst the herbal compounds identified, the andrographolide from Andrographis paniculata [Burm. f.] Nees. strongly induced DnaJB4/ Hlj1 expression via JunB activation and suppressed tumorigenesis both in vitro and in vivo. Andrographolide-induced JunB activation has been shown to modulate AP-2α binding at the MMP2 promoter thereby blocking the expression of MMP-2 408. Additionally, silencing of DNAJB4/HLJ1 partially reversed the blockage of cancer-cell invasion by andrographolide, rendering andrographolide a promising novel anti-tumor agent capable of suppressing tumor growth and invasion in NSCLC.

There is increasing evidence that HSPs may function as immunomodulatory factors. Several investigations within the past years revealed that immunoreactivity of T cells to various types of self-HSPs exerts regulatory properties 409, 410, 411. The regulatory role of DNAJs/HSP40s led to the suggestion that they might function as potential targets for the development of novel anti-inflammatory therapies 15, 235. Furthermore, a peptide derived from the J-domain of DnaJ has been successfully applied in clinical trials of immunotherapy in RA patients 412.