Regulation of p53 and Cancer Signaling by Heat Shock Protein 40/J-Domain Protein Family Members

Heat shock proteins (HSPs) are molecular chaperones that assist diverse cellular activities including protein folding, intracellular transportation, assembly or disassembly of protein complexes, and stabilization or degradation of misfolded or aggregated proteins. HSP40, also known as J-domain proteins (JDPs), is the largest family with over fifty members and contains highly conserved J domains responsible for binding to HSP70 and stimulation of the ATPase activity as a co-chaperone. Tumor suppressor p53 (p53), the most frequently mutated gene in human cancers, is one of the proteins that functionally interact with HSP40/JDPs. The majority of p53 mutations are missense mutations, resulting in acquirement of unexpected oncogenic activities, referred to as gain of function (GOF), in addition to loss of the tumor suppressive function. Moreover, stability and levels of wild-type p53 (wtp53) and mutant p53 (mutp53) are crucial for their tumor suppressive and oncogenic activities, respectively. However, the regulatory mechanisms of wtp53 and mutp53 are not fully understood. Accumulating reports demonstrate regulation of wtp53 and mutp53 levels and/or activities by HSP40/JDPs. Here, we summarize updated knowledge related to the link of HSP40/JDPs with p53 and cancer signaling to improve our understanding of the regulation of tumor suppressive wtp53 and oncogenic mutp53 GOF activities.


Introduction
Tumor suppressor p53 (p53) is a transcription factor that regulates the expression of genes involved in cell cycle arrest and apoptosis, thereby functioning as a tumor suppressor [1,2]. Under non-stressed conditions, the level and activity of wild-type p53 (wtp53) are tightly regulated at a low level mainly through its degradation by the E3 ubiquitin ligase MDM2. Upon genotoxic stresses, p53 protein is post-translationally modified by phosphorylation and acetylation to be stabilized and transcriptionally activated, leading to cell cycle arrest, senescence, and DNA repair for cell survival or apoptosis for cell death [3][4][5][6][7]. While p53 protein mainly localizes to the nucleus, p53 is also detected in the cytoplasm, endoplasmic reticulum (ER), and mitochondria, thereby contributing to a variety of cellular activities [8][9][10][11][12]. Thus, wtp53 prevents cells from undergoing tumorigenesis and is hence called the guardian of the genome [2]. Mutations in the p53 gene are one of the most frequent events in human cancers [13]. The majority of p53 mutations are missense mutations with single amino acid changes in the DNA binding domain, resulting in the production of mutant p53 (mutp53) proteins. Mutp53 is roughly classified into two types, class I and class II, according to the sites of mutations [14]. Class I is a DNA contact type, in which a mutation occurs in amino acids that directly bind to the p53-responsive elements in DNA to impair the p53's sequence-specific DNA binding activity without robust changes in the protein structure. Class II is a structural or conformational type, in which a mutation Increasing evidence indicates that HSP40/JDPs play roles in tumor suppression or progression, and some of them have been shown to regulate the levels and activities of wtp53 and mutp53. Intriguingly, the consequences of interactions between HSP40/JDPs and p53 are varied. A recent review article describes the effects of molecular chaperones on the wtp53 and mutp53 functions [25]. However, there is no review article summarizing the functional association of HSP40/JDPs with p53 and cancer signaling. Here, we focus on compiling reports related to the regulation of p53 activities and cancer progression by HSP40/JDPs, which may help design novel and appropriate targeted cancer therapies.

Class A HSP40/JDPs (DNAJA Proteins)
The class A HSP40/JDPs include DNAJA1, DNAJA2, DNAJA3, and DNAJA4 in mammals [37]. The roles of DNAJA1/HDJ2 and DNAJA3/Tid1 in cancer progression and regulation of p53 function are relatively well documented and are summarized below. DNAJA2 is implicated in cystic fibrosis and neurodegenerative diseases, mainly through degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) and inhibition of tau aggregation; however, its link to cancer development and p53 remains obscure [46][47][48]. Intriguingly, reduced mRNA expression of DNAJA4 by gene methylation is correlated with poor disease-free survival in stomach adenocarcinoma [49]. The hypermethylation status of DNAJA4 is also observed in pediatric embryonal and alveolar rhabdomyosarcoma [50]. However, little is known about the association of DNAJA4 with p53.
• DNAJA1/HDJ2 and cancer DNAJA1/HDJ2 is also implicated in progression of multiple types of cancer [55][56][57][58]. A study suggests the tumor suppressive activity, in which knockdown of DNAJA1/HDJ2 promotes spheroid formation, migration, and invasion of C6 rat glioblastoma cells and reduces survival of rats bearing C6 tumor xenografts [59]. However, most studies indicate the oncogenic role of DNAJA1/HDJ2. High DNAJA1/HDJ2 mRNA expression is associated with poor survival in patients with breast cancer, while DNAJA1/HDJ2 promotes an antiapoptotic phenotype and invasiveness of pancreatic ductal adenocarcinoma cells [60,61]. DNAJA1/HDJ2 also promotes tumor growth and metastasis in human colorectal cancer (CRC) cell lines by interacting with and stabilizing cell division cycle 45 (CDC45) [56]. DNAJA1/HDJ2 also binds to an enzyme involved in extracellular matrix cross-linking and remodeling, transglutaminase 2 (TG2) that is associated with cell survival and cancer progression [62]. The anti-apoptotic activity of DNAJA1/HDJ2 is furthermore supported by a study showing that a DNAJA1-HSP70 complex inhibits nitric oxide-induced CHOPmediated apoptosis, in which farnesylated DNAJA1/HDJ2 binds to BAX and inhibits the translocation of BAX to mitochondria [63]. However, whether p53 is involved in the aforementioned tumor suppressive or oncogenic functions of DNAJA1/HDJ2 has not been investigated.
UD [76] DNAJA1/HDJ2's transcription can be regulated by p53. An algorithm-based study predicts the presence of p53-responsive elements in the human DNAJA1/HDJ2's promoter, which is confirmed by chromatin-immunoprecipitation studies using MCF7 cells (wtp53) [77]. Moreover, a recent report shows that wtp53 indirectly represses mRNA expression of DNAJA1/HDJ2 by inhibiting phosphorylation of heat-shock factor 1 (HSF1), the master regulator of the proteotoxic stress response [78]. However, DNAJA1/HDJ2 levels are unchanged following exogenous introduction of wtp53 in mutp53-knockout HN31 cells [65]. Thus, regulation of DNAJA1/HDJ2 expression by p53 may be dependent on the experimental setting or cellular context.

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DNAJA1/HDJ2 as a cancer therapeutic target Given the cancer-promoting role of DNAJA1/HDJ2, DNAJA1/HDJ2 can be a potential target for cancer therapy. Moreover, DNAJA1/HDJ2 is shown to confer radio-resistance in human SF763 glioblastoma cells (p53 R158L ) [67]. By screening the NCI-approved oncology drugs collection in human chronic myelogenous leukemia HAP1 cell line with or without DNAJA1/HDJ2 knockout, 41 compounds, including cabozantinib, clofarabine, and vinblastine, are identified as drugs that show synergy with DNAJA1/HDJ2 loss [79]. While neither of these studies examines the mutp53 dependency of the radio-and chemotherapyresistance, inhibition or depletion of DNAJA1/HDJ2 may increase the therapy efficacy. Since mutp53 increases radio-and chemotherapy-resistance [80][81][82][83], treatments of cancer cells carrying misfolded/conformational mutp53 with a DNAJA1/HDJ2 inhibitor may effectively increase the sensitivity to radio-and chemotherapies. Currently, no DNAJA1/HDJ2 inhibitor is clinically available, although 116-9e inhibits the DNAJA1/HDJ2 and HSP70 binding, and a chalcone compound, C86, appears to bind to and inhibit several HSP40/JDPs [84,85]. Identifying a compound that specifically inhibits DNAJA1/HDJ2 or multiple HSP40/JDPs would accelerate the development of targeted cancer therapy, specifically for cancers expressing misfolded/conformational mutp53.
• DNAJA3/Tid1 and cancer DNAJA3/Tid1 is implicated in cancer development. In head and neck cancers, high DNAJA3/Tid1 protein levels are correlated with favorable outcome with reduced malignancy and recurrence by inhibiting the galectin-7-TCF3-MMP9 axis or inhibiting the activities of EGFR and AKT [88,98]. Moreover, immunohistochemistry studies using breast cancer tissues show that DNAJA3/Tid1 levels are inversely correlated with tumor malignancy and ErbB2 levels through direct interaction with ErbB2 to promote CHIP-mediated proteasomal degradation [99]. In lung adenocarcinoma, reduced DNAJA3/Tid1 protein levels are correlated with poor overall survival and increased EGFR levels [100]. A recent study in human hepatocellular carcinoma shows that reduced DNAJA3/Tid1 protein levels are associated with increased Nrf2 protein levels and colony-forming potential of human HCC cells, as well as poor clinical outcomes after surgery [101]. In human gastric cancer, reduced DNAJA3/Tid1 expression is correlated with a poor prognosis and increased lymph node invasion in patients. Indeed, knockdown of DNAJA3/Tid1 in gastric cancer cells increases cell migration and invasion with increased protein stability of galectin-7 [86]. Additionally, DNAJA3/Tid1 is shown to interact with von Hippel-Lindau (VHL) protein to induce degradation of HIF-1α, leading to inhibition of VEGF expression and angiogenesis [102]. These observations support tumor suppressive functions of DNAJA3/Tid1. On the other hand, there are a few reports suggesting the oncogenic function of DNAJA3/Tid1. In CRC, increased DNAJA3/Tid1 levels are correlated with colon cancer progression [103].
In non-small cell lung carcinoma (NSCLC), Tid1-S, but not Tid1-L, is required for the EGF-stimulated EGFR transportation into mitochondria, potentially leading to enhanced cancer cell migration and invasion. Indeed, high levels of Tid1-S and EGFR in the mitochondria are correlated with lymph node metastasis and poor overall survival of NSCLC patients [104]. Thus, the roles of DNAJA3/Tid1 in cancer suppression or progression appear to be dependent on the type of cancer and the presence of the variants. However, whether the tumor suppressive or oncogenic functions of DNAJA3/Tid1 are dependent on p53 remains unclear.
• DNAJA3/Tid1 and p53 p53 is another binding partner of DNAJA3/Tid1 (Table 1). Upon hypoxic and genotoxic stress, DNAJA3/Tid1 binds to p53 through the J domain, promoting p53 mitochondrial localization and transcription-independent apoptosis in multiple cancer cell lines [68,69]. Intriguingly, overexpression of DNAJA3/Tid1 enhances mitochondrial translocation of multiple mutp53 (R175H, L194F, R273H, E285K) in several breast cancer and glioblastoma cell lines, resulting in increased mitochondrial apoptosis regardless of the presence of hypoxic stress [68]. Thus, DNAJA3/Tid1's binding to mutp53 may restore the transcription-independent mitochondrial apoptotic function of p53. Hence, increasing the DNAJA3/Tid1 levels or activity could be used as a strategy to induce apoptosis in p53-mutated cancers.
• DNAJB1/HDJ1 and cancer DNAJB1/HDJ1 is also implicated in cancer progression and therapeutic resistance [24,132,133]. A fusion gene of DNAJB1/HDJ1 and PRKACA (protein kinase cAMP-activated catalytic subunit alpha), resulting from an~400 kb of in-frame deletion on chromosome 19, is found in nearly all cases of fibrolamellar hepatocellular carcinoma (FL-HCC) [115]. As a mechanism, the DNAJB1-PRKACA fusion protein accelerates the FL-HCC tumorigenesis by cooperating with the WNT pathway [133]. Moreover, DNAJB1/HDJ1 binds to mitogen-inducible gene-6 (MIG6), a tumor suppressor that inhibits the EGFR signaling, which decreases the protein level of MIG6 by enhancing its ubiquitination, leading to upregulation of the EGFR signaling pathway in A549 lung adenocarcinoma and HCT116 CRC cell lines [132,134]. Additionally, DNAJB1/HDJ1 is identified as a biomarker for cholangiocarcinoma [135]. Thus, DNAJB1/HDJ1 promotes the progression of multiple types of cancer, although it remains to be determined whether p53 is involved in these oncogenic activities of DNAJB1/HDJ1.

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DNAJB1/HDJ1 and p53 DNAJB1/HDJ1 binds to and regulates the activities of both wtp53 and mutp53 (Table 1). An in vitro study using purified proteins reveals that DNAJB1/HDJ1 forms a complex with p53 (both wtp53 and p53 R175H ) in the presence of HSC70 and ATP [136]. In support of this finding, Sugito et al. [137] observe the intracellular complex of p53 Y205C , DNAJB1/HDJ1, and HSP70 in human oral squamous cell carcinoma HOC815 cells. DNAJB1/HDJ1 appears to contribute to tumor suppression via regulation of the wtp53 activity. Specifically, DNAJB1/HDJ1 binds to and stabilizes MDM2, a major ubiquitin ligase of p53; however, this interaction inhibits MDM2's activity on p53, leading to p53 activation, while DNAJB1/HDJ1 knockdown increases cell proliferation and tumor growth of MCF7 cells in a manner dependent on wtp53 [41]. It should be noted that the C-terminal region of DNAJB1/HDJ1, lacking the J domain, is sufficient for interacting with and stabilizing MDM2, suggesting that DNAJB1-mediated stabilization of MDM2 is likely HSP70 independent. Silva et al. [138] also observe that DNAJB1/HDJ1, whose mRNA and protein levels are induced by trans-chalcone (TChal), binds to wtp53, leading to stabilization and activation of wtp53 in U2OS cells. These findings suggest the tumor suppressive role of DNAJB1/HDJ1. However, Cui et al. [70] show that DNAJB1/HDJ1 binds to and induces degradation of Programmed Cell Death 5 (PDCD5), a positive regulator of p53-mediated apoptosis, in HCT116 cells, while knockdown of DNAJB1/HDJ1 enhances etoposide-mediated inhibition of colony formation with an increase in the PDCD5 levels and cell death in A549 cells. These results suggest that DNAJB1/HDJ1 inhibits wtp53's apoptotic function through interaction with PDCD5 and contributes to cancer progression. Thus, the effects of DNAJB1/HDJ1 on the wtp53 activity are dependent on the cellular context or experimental settings.
Some studies have examined the functional interaction between DNAJB1/HDJ1 and oncogenic mutp53. Specifically, DNAJB1/HDJ1, together with HSP70, appears to facilitate binding of conformational mutp53 (R175H) with TAp73, a p53 family member. This complex induces chemoresistance to several DNA damaging reagents. Intriguingly, when MDM2 is overexpressed in cells carrying mutp53, this complex is inhibited by MDM2, which switches to the formation of mutp53-TAp73-MDM2 complex, leading to furthermore enhanced resistance to cisplatin, etoposide, and doxorubicin in SkBr3 and H1299 cells expressing p53 R175H [24]. This could explain why breast cancer patients with p53 mutations and high levels of MDM2 show poorer overall survival than those with p53 mutations or MDM2 overexpression alone [24]. The observation that DNAJB1/HDJ1 supports mutp53's oncogenic GOF is also supported by a report by Parrales et al. [23] in which DNAJB1/HDJ1 contributes to the accumulation of p53 R175H .
Intriguingly, Hiraki et al. [71] identify a natural compound, chetomin (CTM) as a compound that enhances the interaction of DNAJB1/HDJ1 with p53 R175H to restore the wtp53-like activity. CTM inhibits cell proliferation and tumor growth specifically in TOV-112D and CAL33 cells carrying p53 R175H with mRNA upregulation of p53 target genes, p21, PUMA, and MDM2, and increased DNA binding activity of p53 R175H to p53-responsible elements in these genes. Thus, the interaction of DNAJB1/HDJ1 with p53 R175H induced by CTM may promote refolding of p53 R175H , rather than enhancing mutp53 GOF activity or stability as observed by other reports [23,24]. Since these experiments are not tested using cells lacking DNAJB1/HDJ1, it remains unclear whether the biological activity and mutp53 reactivation by CTM are solely dependent on DNAJB1/HDJ1. The discrepancies may be caused by the unappreciated function of CTM or differences in the cellular context. Together, whether DNAJB1/HDJ1 functions as a tumor suppressor or an oncogene appears to be dependent on the presence of wtp53 or mutp53, experimental settings, and the cellular context.
• DNAJB9/MDG1/ERdj41 and cancer DNAJB9/MDG1/ERdj4 also plays a role in tumor suppression. In breast cancers, the DNAJB9/MDG1/ERdj4 mRNA level is lower than that in normal breast tissues, which is correlated with poor clinical outcomes [144]. Moreover, DNAJB9/MDG1/ERdj4 binds to and stabilizes F box/SPRY domain-containing protein 1 (FBXO45) to promote FBXO45-mediated ubiquitination and degradation of zinc finger E-Box binding homeobox 1 (ZEB1), leading to inhibition of migratory, invasion, and in vivo metastases of MDA-MB-231 cells [144]. However, involvement of p53 in these phenotypes has not been investigated.
• DNAJB9/MDG1/ERdj41 and p53 DNAJB9/MDG1/ERdj4 also interacts with wtp53 (Table 1). Lee et al. [72] show that DNAJB9/MDG1/ERdj4, whose mRNA expression is indirectly induced by wtp53, binds to wtp53 and inhibits p53-mediated apoptosis under genotoxic stress. While a DNAJB9/MDG1/ERdj4 mutant lacking the J domain cannot bind to wtp53, whether HSP70 is involved in the observed p53 inhibition by DNAJB9/MDG1/ERdj4 needs to be clarified. The same group also shows that DNAJB9/MDG1/ERdj4 overexpression inhibits the H-RAS V12 -induced p53-dependent senescence in MEFs and promotes the cellular transformation [73]. In support of these findings, in human non-gestational choriocarcinoma samples, two missense mutations in the DNAJB9/MDG1/ERdj4 gene are found (F46Y, H47R), while the introduction of site-specific mutations in the DNAJB9/MDG1/ERdj4 gene in gestational choriocarcinoma JEG-3 cells results in reduced DNAJB9/MDG1/ERdj4 mRNA and protein levels with increase in the wtp53 levels [74]. These studies suggest the tumor-promoting function of DNAJB9/MDG1/ERdj4 by inhibiting wtp53, which is distinct from the aforementioned tumor-suppressive function of DNAJB9/MDG1/ERdj4 in breast cancer by Kim et al. [144]. Thus, the roles of DNAJB9/MDG1/ERdj4 in cancer suppression or progression could be dependent on the presence of wtp53 in cells or other cellular contexts.

Other DNAJB Members and Cancer
DNAJB4/HLJ1 is not only implicated in myocardial infarction and Alzheimer's disease [145,146], but also regulates cancer progression. In NSCLC and CRC, DNAJB4/HLJ1 levels are inversely correlated with clinical outcomes [147,148]. In invasive breast carcinoma, the DNAJB4/HLJ1 level is significantly lower, as compared to normal breast tissues, benign neoplasm, and ductal carcinoma in situ [149]. Tsai et al. [147] also show that exogenous expression of DNAJB4/HLJ1 reduces invasion, migration, proliferation, colony formation, and primary tumor growth of lung adenocarcinoma cells with increasing p21 levels; however, these phenotypes are p53-independent [147]. Overall, DNAJB4/HLJ1 functions as a tumor suppressor, but its functional relationship with p53 remains unclear.
• DNAJB6/MRJ DNAJB6/MRJ is another DNAJB member implicated in cancers. High levels of DNAJB6/MRJ are associated with poor outcomes in patients with CRC, while knockdown of DNAJB6/MRJ in HCT116 and SW480 CRC cells inhibits invasion and pulmonary metastases with reduced IQ Motif Containing GTPase Activating Protein 1 (IQGAP1) levels, a scaffold protein of the MAP kinase pathway [123]. Lin et al. [150] also show that DNAJB6/MRJ promotes cell adhesion, migration, and invasion through stabilizing uPAR, as well as phosphorylation of FAK, ERK1/2, and AKT, in HCT116 cells. These reports suggest oncogenic roles of DNAJB6/MRJ. However, in breast cancer cells, DNAJB6/MRJ inhibits cancer progression by binding to HSPA8 and inhibiting Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT) [120,151,152]. Moreover, in esophageal squamous cell carcinoma (ESCC), nuclear localization of DNAJB6/MRJ is associated with favorable outcomes in patients with ESCC, while DNAJB6a, an isoform that contains a nuclear localization signal, reduces proliferation and xenograft tumor growth with reduced AKT signaling [122]. Thus, the function of DNAJB6/MRJ may be dependent on tissue type or the presence of the isoforms. Whether DNAJB6/MRJ has any impact on p53 activity needs to be determined as a future study.
• DNAJB8 DNAJB8 functions to protect against protein toxicity associated with polyQ aggregation diseases [153,154]. DNAJB8 is also identified as a factor that induces cancer stem-like properties, such as tumor-initiating ability and drug resistance in human CRC and renal cell carcinoma (RCC) cell lines [124,155,156]. However, the underlying mechanisms and the potential involvement of p53 remain to be elucidated.

Class C HSP40/JDPs (DNAJC Proteins)
Any HSP40/JDPs, which do not belong to either class A or B, are categorized into class C. The class C HSP40/JDPs are comprised of at least 32 members with the greatest diversity in their molecular sizes, structures, and functions [37,164]. While DNAJC proteins are implicated in a variety of diseases, limited studies have shown their roles in cancer progression [165,166]. Of 32 DNAJC members, DNAJC12, DNAJC15/MCJ, and DNAJC25 are implicated in cancer progression, while members linked to p53 include DNAJC2/ZRF1, DNAJC7/TPR2, and DNAJC9.
On the other hand, DNAJC2/ZRF1 is overexpressed in human acute myelocytic leukemia (AML), and depletion of DNAJC2/ZRF1 results in decreased cell proliferation with increased apoptosis and cell differentiation, thus showing oncogenic function [172]. This may be caused, at least partially, by its binding to retinoic acid receptor α (RARα), since DNAJC2/ZRF1 knockdown enhances RA-mediated suppression of HL60 xenografts [172]. DNAJC2/ZRF1 protein levels are also upregulated in gastric cancer tissues as compared to non-tumor tissues, which is correlated with poor overall outcomes [75].
• DNAJC2/ZRF1 and p53 DNAJC2/ZRF1's oncogenic function may be dependent on p53. Knockdown of DNAJC2/ZRF1 in human gastric cancer cell lines inhibits cell proliferation and migration and induces apoptosis with increased p21 levels, especially when cells carry wtp53 (Table 1). These phenotypes are minimally observed in cancer cells with p53 mutations and p53 null [75], suggesting that DNAJC2/ZRF1 inhibits the function of wtp53 to promote cancer progression. Thus, DNAJC2/ZRF1 acts as either a tumor suppressor or an oncogene, depending on the cellular context, including the cancer type and/or the presence of wtp53 in cells.
• DNAJC7/TPR2 and cancer DNAJC7/TPR2 is also implicated in cancer progression. Increased polyglutamylated-DNAJC7/TPR2 levels in sera may serve as a potential biomarker for early detection of RCC and are also associated with advanced stage and grade of RCC [180]. However, the underlying mechanism remains unclear.
• DNAJC7/TPR2 and p53 Kubo et al. [76] identify DNAJC7/TPR2 as a regulator of p53. DNAJC7/TPR2 binds to the DNA binding domain of p53. This interaction stabilizes and activates p53, leading to inhibition of the colony-forming potential of H1299 cells exogenously expressing wtp53, with increased p21, BAX, and MDM2 mRNA levels. Future studies are required to determine whether the tumor suppressive function of DNAJC7/TPR2 is entirely dependent on p53 or if DNAJC7/TPR2 could show any oncogenic functions in different experimental settings or cellular contexts.

DNAJC9
DNAJC9 activates the HSP70's ATPase activity through the J domain as other HSP40/JDPs. DNAJC9 mRNA and protein levels are upregulated by various stress and mitogenic stimuli [181]. DNAJC9 mainly localizes to the nucleus; however, upon heat shock stress, DNAJC9 is exported to the cytoplasm and plasma membrane [181]. Recently, Hammond et al. [182] demonstrate that DNAJC9 forms a complex with a DNA replication licensing factor MCM2 and a histone H3-H4 dimer to recruit multiple HSP70 enzymes and fold histone H3-H4 dimers, for maintenance of a proper supply of histones during active replication and transcription. Intriguingly, DNAJC9 is implicated in familial recurrent corneal erosion dystrophy, epithelial recurrent erosion dystrophy, and schizophrenia [183,184].
• DNAJC9 and cancer DNAJC9 is also implicated in cancer progression. DNAJC9 is upregulated in basal, HER2, and luminal B breast cancers, as well as in node-positive cervical squamous cell carcinoma [185]. By comprehensive analysis of transcriptomic profiles of HSP family genes in 9018 patients with 28 cancers, Liu et al. [186] show that DNAJC9 mRNA is upregulated in multiple different cancer types with prognostic values. These include adrenocortical carcinoma, AML, prostate adenocarcinoma, and lung adenocarcinoma. However, how DNAJC9 contributes to cancer progression remains to be elucidated.

• DNAJC9 and p53
While there is no report showing the regulation of p53 activity by DNAJC9, DNAJC9 appears to be a downstream target of p53 in the zebrafish system; an in silico screening of the zebrafish genome identifies a p53-responsive element in the zebrafish DNAJC9 gene [187]. The chromatin-immunoprecipitation and luciferase reporter assays further confirm the interaction of p53 with the promoter region of the zebrafish DNAJC9 gene.

Other DNAJC Members and Cancer
• DNAJC12 DNAJC12 is implicated in dystonia and intellectual disability [188], mild hyperphenylalaninemia [188][189][190], and Parkinson disease in the Chinese Han population [191]. Some oncogenic roles of DNAJC12 have also been reported. In rectal cancer, increased DNAJC12 levels are correlated with poor chemotherapy response [192]. In gastric cancer, increased mRNA expression of DNAJC12 is correlated with cancer invasion, lymph node metastasis, and disease progression, thus having higher morbidity and mortality rates [193]. Moreover, in lung cancer, DNAJC12 levels are upregulated, while DNAJC12 knockdown reduces the malignant properties and tumor growth of lung cancer cells in vitro and in vivo by inhibiting activation of β-catenin [194]. However, none of these studies address if there is a link with p53.
DNAJC15/MCJ is a unique member that localizes at the mitochondrial inner membrane. DNAJC15/MCJ functions as a negative regulator of the respiratory chain and inhibits the complex I activity and mitochondrial membrane potential, promoting ROS overproduction and ATP depletion [195]. DNAJC15/MCJ potentially acts as a tumor suppressor by promoting the release of pro-apoptotic molecules through the mitochondrial permeability transition pore complex [196]. Indeed, DNAJC15/MCJ is frequently hyper-methylated in multiple types of human cancer, including malignant pediatric tumors, neuroblastoma, Wilm's tumor, melanoma, and breast cancer [197][198][199][200]. Reduced expression of DNAJC15/MCJ is also correlated with increased drug resistance, as well as increased levels of c-JUN protein and its downstream target ATP binding cassette subfamily B member 1 (ABCB1)/multiple drug resistance 1 (MDR1), in ovarian and breast cancers [200][201][202][203]. Whether p53 is involved in the tumor suppressive function of DNAJC15/MCJ has not been investigated.
• DNAJC25 DNAJC25 is relatively a new member of the HSP40/JDP subfamily C, and hence little is known about the function of this protein. In liver cancers, DNAJC25 mRNA expression is markedly reduced, while its overexpression induces apoptosis and inhibits colony formation of liver cancer cells [204]. In silico analyses suggest that DNAJC25 mRNA expression is also reduced in breast cancer tissues, and high DNAJC25 mRNA expression is correlated with favorable post-progression survival in breast cancer [205]. These results suggest the tumor suppressive role of DNAJC25, but the functional association of DNAJC25 with p53 remains to be elucidated.

Discussion
Both wtp53 and mutp53 proteins can be misfolded under a variety of cellular conditions (e.g., heat shock, genotoxic stress) like other proteins. These misfolded proteins as well as properly folded native proteins are detected by molecular chaperone systems, including HSPs, to be refolded, stabilized, or degraded. Indeed, both wtp53 and mutp53 are bound to and functionally regulated by the HSP system [206][207][208][209][210][211]. Since the HSP system has a great impact on the protein levels and functions, understanding the mechanisms by which HSPs detect and regulate the structure and functions of wtp53 and mutp53 would help design efficient p53-targeted anti-cancer therapies. At least fifty members of HSP40/JDPs are present in cells, and each has different clients [37]. This may explain the diverse regulation of target proteins including p53 by HSP40/JDPs and different biological outcomes. Several literatures suggest HSP70-independent functions of HSP40/JDPs in in vitro and in vivo [39,40,[42][43][44][45]. Hence, whether the oncogenic or tumor suppressive functions of each HSP40/JDP are HSP70-dependent or -independent needs to be clarified in the future.
Recent studies suggest that wtp53 acts like mutp53 under specific conditions, referred to as pseudo-mutant p53. Indeed, previous studies indicate that wtp53 could be misfolded and form a mutant conformation, especially in the absence of specific chaperones, leading to reduced wtp53 transcriptional activity and cancer progression [212][213][214]. To support this finding, Arandkar et al. [215] observe that wtp53 in cancer-associated fibroblasts (CAFs), but not in normal fibroblasts, acquires misfolded conformation and rather enhances migration, invasion, and tumor growth of lung cancer cells. Moreover, a small subset of breast cancers with wtp53 display gene expression patterns similar to those carrying mutp53, including upregulation of genes specifically regulated by mutp53 (e.g., PSAT1, TAP1, AurkA, CDC45, MAD2L1, ATL3) [216]. Additionally, in a subpopulation of preleukemic hematopoietic stem/progenitor cells from primary human AML that carry DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) mutations, high levels of pseudo-mutant p53 are dominantly detected over the wild-type conformation, while p53 in leukemic blasts shows mainly the wtp53 conformation [217]. It remains unknown how and under which conditions wtp53 conformation converts to mutant conformation and whether the process is reversible. Given the role of HSP40s/JDPs in protein folding and refolding, some members of HSP40s/JDPs could be involved in this process.
Understanding the mechanism of p53 regulation by HSP40/JDPs is crucial for developing novel p53-targeted therapies, given that current wtp53-and mutp53-targeting therapies have not yet been successful. Increasing the wtp53 activity specifically in tumors is required for successful targeted cancer therapy. For mutp53-expressing cancers, depletion or reactivation of mutp53 should be achieved without activating wtp53 in non-tumor cells. Given that HSP40/JDPs play central roles in the stabilization or degradation of wtp53 and mutp53, as well as reactivation of mutp53, controlling the p53 (wtp53, mutp53) levels and activity via HSP40/JDPs could be an alternative strategy for cancer therapy, instead of directly targeting p53.

Conclusions
We have given an overview of HSP40/JDPs mainly by focusing on their roles in cancer signaling and p53 functions ( Figure 2 and Table 1). HSP40/JDPs detect misfolded structures of proteins and alter their localization, stability, or functions, therefore involved in numerous diseases including cancers. The activities of wtp53 and mutp53 can be fine-tuned by multiple HSP40s/JDPs, partially explaining diverse p53 functions in various cellular contexts and tumor microenvironments. Until now, only six HSP40/JDPs (DNAJA1, A3, B1, B9, C2, C7) have been found to regulate wtp53 and/or mutp53 activities, though 16 members are implicated in cancer progression. Identifying more HSP40/JDPs involved in cancer progression and p53 (wtp53, mutp53) activities, as well as the underlying mechanisms, will advance our knowledge of cancer progression and may accelerate the development of novel anti-cancer therapies.