Introduction

Ferruccio Ritossa in 1962 first discovered Heat shock proteins (HsP) or stress proteins (Ritossa 1962). He observed temperature induced puffing patterns in salivary gland chromosomes of Drosophila melanogaster larvae. The increase in temperature stimulated the expression of proteins with molecular masses of 26 and 70 kDa (Tissieres et al. 1974). As such, the original definition of HsP was based on their enhanced expression in response to cellular insults, such as raised temperature, oxidative stress, chemical exposure and irradiation (Young et al. 2004). Under normal physiological conditions, a complete set of functionally competent proteins are maintained in the cell. When exposed to cellular stressors, disturbance of the intracellular milieu induces a stress response in the cell, which inhibits the activity of many housekeeping genes while activating stress genes (Gao et al. 2004). This leads to increased levels of stress protein and their chaperones in the cell in a concerted effort to maintain protein homeostasis.

Chaperoning System

In 2008 the ‘chaperoning system’ has been projected as a concept to include all molecular chaperones, co-chaperones, and cofactors of an organism (Macario and Conway de Macario 2009). The concept visualizes a physiological system encompassing all chaperones and their functionally-related molecules and structures, in all tissues, organs, and biological fluids. This physiological system is essential for the control of protein homeostasis and maintenance of a complete set of proteins in all fluids, cells, and tissues, with the correct and functional conformation (Macario and Conway de Macario 2009). Many vital biological processes such as antigen presentation, hormone receptor assembly, formation of complexes with a variety of ligands and unrelated to protein homeostasis also show direct involvement of the chaperoning system (Macario et al. 2010). The scientific discipline that deals with the chaperoning system is called chaperonology which also includes the study of the genomic sequences of chaperone genes (e.g. by applying chaperonomics) (Brocchieri et al. 2007), the study of the diseases that involve chaperones as causative factors (i.e.chaperonopathies) (Macario and Conway de Macario 2005), and the use of chaperones (molecules and genes) for the treatment of chaperonopathies (i.e.chaperonotherapy) (Macario et al. 2010).

Classification of Heat Shock Proteins

Hsps are classified into six major families according to their molecular mass in kilodaltons (Schlesinger 1990) (a) the large molecular weight Hsp of100–110 kDa, (b) the Hsp90 family of 83–90 kDa, (c) the Hsp70 family of 66–78 kDa, (d) the Hsp60 family of 55–64 kDa, (e) the Hsp40 family of 35–54 kDa, and (f) the small Hsp of 8–34 kDa. The family members have functional homologs in different compartments of the cell. There is a high degree of homology between the Hsp counterparts of different organisms (e.g., the Escherihia coli DnaK and the human Hsp70 share approximately 50% sequence identity). However, no noticeable sequence homology between different families of Hsp could be perceived (e.g., Hsp60 and Hsp70).

The Hsp70 system collaborates with extended peptide segments of proteins as well as partially folded proteins to avert aggregation, modification of folding pathways, and modulation of activity. When not in a state of interaction with a substrate peptide, Hsp70 is typically in an ATP bound state. A very weak intrinsic ATPase activity is possessed by Hsp70. As newly synthesized proteins emanate from the ribosomes, the substrate binding domain of Hsp70 identifies sequences of hydrophobic amino acid residues, and interacts with them. After a peptide binds on the binding domain of Hsp 70, ATPase activity of Hsp70 is stimulated and the slow rate of ATP hydrolysis is enhanced. On ATP hydrolysis to ADP, the binding pocket of Hsp70 closes, thus, the now-trapped peptide chain is tightly bound (Mashaghi et al. 2016).

Chaperones Act in Multi Chaperone Complexes

Although chaperones are relatively abundant, they very rarely function alone (Smith et al. 1995). They typically create large multiprotein complexes that contain other chaperones, co chaperones and various accessory proteins. Chaperone assisted folding is a complex multistep process based on non-covalent interactions between chaperones and their substrates, called “clients”. The folding cycle of Hsp90 is propelled by ATP hydrolysis which aids conformational changes and the recruitment of different co-chaperones. The mechanism of the Hsp90 folding cycle was described for the maturation of steroid-hormone receptors (SHR) by Smith et al. (Smith et al. 1995). The chaperone cycle starts when the newly synthesized or denatured client protein associates with Hsp70 (heat-shock protein of 70 kDa), Hsp40 (heat-shock protein of 40 kDa) and the adapter HIP (Hsp70-interacting protein) to form an early complex. Then adapter protein HOP (Hsp70/Hsp90-organising protein), that binds both Hsp70 and Hsp90 chaperones simultaneously, shifts the client protein to Hsp90 dimer and displaces Hsp40 to form an intermediate complex. In an ATP-dependent manner, the Hsp90 dimer binds the client protein and Hsp70, HOP and HIP are replaced by cochaperones p23 and CYP40 (cyclophilin 40) to complete the mature complex. Hormone binding to SHR in the mature complex leads to a conformational change of SHR driven by ATP hydrolysis. Finally, SHR is dissociated and transferred to the nucleus to regulate gene transcription. The spectrum of folded clients is also influenced by association of Hsp90 with different co-chaperones. For example, Cdc37 (cell division cycle 37) is a co-chaperone which binds to the N-terminal domain of Hsp90 and facilitates the recruitment of various kinases to the Hsp90 machinery (Smith et al. 1995).

Hsp70 binds tightly to partially synthesized peptide sequences (incomplete proteins), therefore forbidding them from aggregation and being relinquished as nonfunctional. After the synthesis of the entire protein, a nucleotide exchange factor (such as BAG-1 and HspBP1) prompts ADP release and binding of fresh ATP, opening the binding pocket. The protein can fold free on its own, or can be transported to other chaperones for further processing. Peptides can be transferred from Hsp70 to Hsp90 with the help of HOP (Hsp70/Hsp90 Organizing Protein) as HOP can bind to both Hsp70 and Hsp90 concomitantly. Hsp70 also facilitates in transmembrane relocation of proteins, by ossifying them in a partially folded state. Hsp70 proteins can protect cells from thermal or oxidative stress. These stresses normally act to impair proteins, fostering partial unfolding culminating in aggregation. By transiently binding to hydrophobic residues exposed by stress, Hsp70 intercepts these partially denatured proteins from aggregating, and permits them to refold. Low ATP is an attribute of heat shock and strengthened binding is seen as aggregation repression, while recovery from heat shock inculcates substrate binding and nucleotide cycling (Wegele et al. 2004).

Hsp70 is able to take part in disposition of damaged or defective proteins. These altered proteins are directed to the cell’s ubiquitination and proteolysis pathways through interaction with CHIP (Carboxyl-terminus of Hsp70 Interacting Protein) –an E3 ubiquitin ligase (Lüders et al. 2000). Finally, in addition to improving overall protein integrity, Heat-shock protein 70 (Hsp70) can block apoptosis at several levels, and is thought to play a carcinogenic role related to its antiapoptotic activity (Lüders et al. 2000; Murphy 2013). One distinctive feature of apoptosis is the release of cytochrome c, which then employs Apaf-1 and dATP/ATP into an apoptosome complex. This complex then cleaves procaspase-9, leading to caspase-9 activation and eventually induction of apoptosis via caspase-3 activation. The arrival of procaspase 9 to this complex is blocked by Hsp70. It renders procaspase-9 binding less favorable by inducing a conformational change. Hsp70 is shown to interplay with endoplasmic reticulum stress sensor protein IRE1alpha thereby safeguarding the cells from ER stress induced apoptosis. This interaction prolonged the splicing of XBP-1 (a transcription factor) mRNA thereby actuating transcriptional upregulation of targets of spliced rescuing the cells from apoptosis (Gupta et al. 2010). HBP 21, a chaperone of heat shock protein 70, acts as tumor suppressor in hepatocellular carcinoma. But, due to allele loss and aberrant methylation when HBP21 is down regulated, HSP70 confers resistance to apoptosis by blocking Bax translocation to mitochondria (Yang et al. 2012) which is required for cytochrome c release. Thus, HBP21 can promote cell apoptosis caused by unfavorable conditions and exert negative influence on Hepatocellular carcinoma (HCC) pathogenesis.

Chaperones maintain protein homeostasis not only by maturation of newly synthesised proteins and stabilization of unstable proteins, but also by recognitionand transport of defective proteinsto the degradation pathway. This needs the recruitment of another co-chaperone, CHIP (an E3 ubiquitin ligase), into the Hsp90 chaperone machinery (Murata et al. 2001). It was shown that CHIP suppresses tumor progression in human breast cancer by enhancing the degradation of several oncogenic proteins (Kajiro et al. 2009). Moreover, knockdown of CHIP in breast cancer cells results in rapid tumor growth and metastatic phenotypes in mice. The mechanisms regulating the protein folding/degradation balances involve chaperone binding to CHIP and HOP that depends on a phosphorylation state of Hsp90 and Hsp70 C-termini (Muller et al. 2013). The phosphorylation of these chaperones prevents binding to CHIP and enhances binding to HOP. Proliferating cells express lower levels of CHIP and higher levels of HOP, Hsp70 and Hsp90 compared to non-proliferating cells (Ruckova et al. 2012). Decreased CHIP expression in proliferative cells supports its proposed tumor suppressor properties, while overexpression of HOP may contribute to excessive Hsp90 activity and stabilization of client proteins in cancer cells. These reports reflect elevated protein folding environment in cancer cells regulated by the action of co-chaperone expression and chaperone modifications.

Cancer Stem Cells (CSC) in GI Cancer: Role of Hsp70

Our understanding of phenotypic plasticity of gastrointestinal cancer cells has considerably evolved in the past decade (Bekaii-Saab and El-Rayes 2017; Brungs et al. 2016). Evidences from several malignancies indicate that the cancer cells acquire stem-like phenotype often after chemotherapy (Monteiro and Fodde 2010; Hu et al. 2012). This has been substantiated both in functional assays (ALDH1 activity) and by the expression of several stem cell markers (CD44, CD133, NANOG, SOX2, BMI1, ALDH1, cMYC, KLF4 etc. (Hadjimichael et al. 2015). The hypothesis that a small sub-population of the tumor bulk maintains a stem-like phenotype and accounts for the therapeutic refractoriness, dormant behaviour with subsequent metastasis and recurrence has now been experimentally established using genetic lineage tracing of cancer cells in mouse model. CSC is at the hierarchical apex of tumorcells with the potency of self-renewal, clonal growth and differentiation into different cell types which characterizes the tumor bulk (Alcolea and Jones 2013; Kretzschmar and Watt 2012; Schepers et al. 2012).

Gastric CSCs were first described in 2007 by Yang et al. (Yang et al. 2007). The gastric cells that were isolated from patient tumors showed several phenotypic characteristics of stem and progenitor cells as determined by their enhanced clonogenic potential and differentiation capacity, high susceptibility to spontaneous immortalization and the expression of stem cell inversely correlated with patient survival abrogating the expression of GRP78 marker OCT4, and Nanog. Work by Takashi et al. further showed that CD44 can be a reliable cell surface marker for isolating gastric cancer stem cells (Takaishi et al. 2009). Gastric cancer cell lines MKN-45, MKN-74, and NCI-N87 had a significant subpopulation of high CD44 expressing cells which can be FACS sorted into CD44+ and CD44− population. CD44+ cells formed tumor spheres and showed higher tumorigenic ability when injected into stomach and skin of severe combined immunodeficient (SCID) mice. Further, The CD44+ gastric cancer cells differentiated to give rise to CD44(−) cells. CSCs are also critical for the formation and maintenance of liver metastasis derived from colorectal cancers (de Sousa e Melo et al. 2017). The role Hsp70 in the malignant progression of highly metastatic human colorectal cancer (CRC) cell lines suggested a strong correlation with the advanced clinical stages and positive lymph node involvement (Hwang et al. 2003). Particularly in colorectal cancer, CD44–hyaluronan (HA) binding, leads to the assembly of a signalling complex that includes Hsp70 and the co-chaperone CDC37, which promotes phosphorylation and activation of the ERBB2–ERBB3 receptor tyrosine kinases (RTKs). The CD44–ERBB2–ERBB3 complex provides a strong stimulus for cyclooxygenase 2 (COX2) transcription and thus trigger the anti-apoptotic machinery in the CSC population (Misra et al. 2015).

GRP78 (BiP/HSPA5), is an important member of the Hsp70 superfamily that is evolutionarily conserved across a wide range of species from yeast to humans, and plays a crucial role in the maintenance of the embryonic stem cells (Wang et al. 2010). Wu et al. have recently demonstrated a significantly high expression of GRP78 in CSC population of the Head and neck squamous cell cancer (HNSCC) (Wu et al. 2010). Depleting GRP78 reduced the self-renewal ability, expression of stemness genes, tumorigenic potential both in cell based and mouse xenograft models and induced apoptotic cell death, thus providing a potential therapeutic avenue. Studies conducted on 86 cases of resected gastric cancer demonstrated a positive correlation of GRP78 with lymph node metastasis. Compared to the adjacent non tumorigenic region of the gastric mucosa GRP78 was specifically overexpressed in the tumor specimens including both primary tumors and metastatic lymph nodes (Zhang et al. 2006). Although this study did not focus on CSC population of the gastric tumors, but given the elevated expression of GRP78 both in primary and secondary metastatic sites of gastric tumors, and the similarity in phenotypic characteristics observed in HNSCC, a putative role of GRP78 in the gastric cancer can be envisioned and warrants further study.

Most preclinical and clinical studies on gastro-intestinal carcinoma have indicated that maximum therapeutic advantage in terms of tumor regression, inhibition of metastasis and recurrence, along with resenstization of persistent tumors to second line chemotherapy could be achieved by the combinatorial therapy targeting both the non-stem-like differentiated tumor bulk and the CSC population (Takaishi et al. 2009; Vries et al. 2010). In lymphoma, siRNA-mediated attenuation of HSF1 targeted both the CSC and the non CSC population arguing strongly in favor of Hsp90/Hsp70 inhibitors for treating gastrointestinal cancers that are frequently plagued by chemoresistance (Newman et al. 2012). Promising data was obtained using small molecule inhibitors of Hsp70 and Hsp90 in the in vitro studies conducted on colorectal cancer (Massey et al. 2010). Treatment of colon cancer cells with the Hsp70 inhibitor VER-155008 or Hsp90 inhibitor 17AAG alone, did not exhibit any potent therapeutic effect, but a combinatorial therapy induced more than 90% cell death. Similar results were also obtained by combining another Hsp90 inhibitorVER-82,160 with VER-155008 in HCT116 colon carcinoma cell lines (Massey et al. 2010). It is likely that while Hsp90 inhibitor induces a more cytotoxic effect in the non CSC population, ablation of the CSC population is achieved by the inhibition of Hsp70 function (Fig. 1).

Fig. 1
figure 1

A literature extracted representation of chaperoning in pancreatic cancer pathogenesis

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Pancreatic Cancer: An Overview

Pancreatic ductal adenocarcinoma is one of the most aggressive cancers and is the fourth most-frequent tumour-related cause of death in the Western world (Siegel et al. 2012). The prognosis for patients with this disease is extremely poor, with an overall median survival of 5–8 months and fewer than 5% have a long-term survival of more than 5 years (Bilimoria et al. 2007). Although patients who do not undergo surgical resection of the tumor do not have a long prognosis (median survival of about 6 months), the 5-year survival rate after resection plus adjuvant therapy is over 20% (Bilimoria et al. 2007). About 70% of all patients develop metastatic recurrence even after successful surgical resection (Bilimoria et al. 2007; Oettle et al. 2007). Therefore, pancreatic adenocarcinoma is considered to be a systemic disease in the majority of patients at the time of diagnosis. Consequently, adjuvant chemotherapy is the standard of care after surgical resection, and has been shown to further increase long-term survival rates by about 10% (Oettle et al. 2007).

The increased expression of one or more Hsp above the level observed in normal tissues is a common feature of human cancers, both solid tumours and haematological malignancies (Kimura et al. 1993). A number of molecular chaperones have been found to be over expressed in human pancreatic cancer (detailed in the Table 1).

Table 1 Important chaperones and co-chaperones in human pancreatic cancer
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Role of Chaperones in Human Pancreatic Cancer Pathogenesis

This may be an adaptive response by pancreatic cancer cells to maintain protein homeostasis and promote cell survival in an unfavorable environment, as well as to stimulate cell proliferation and inhibit cell death (given in Table 2). Increased amounts of Hsps allow pancreatic cancer cells to tolerate changes from within, such as potentially lethal mutations that have a role in oncogenesis. Chaperones, such as Hsp90, are known to be highly expressed in most tumor cells, including pancreatic cancer cells (Giessrigl et al. 2012). Hsp90 also acts as protectors for genetic aberrations found in cancer, facilitating the performance of mutated proteins in promotion of malignancy while conferring cellular tolerance to the imbalanced signaling produced by these oncoproteins (Zhang et al. 2011). Indeed, Hsp are seen to participate in the six essential alterations in cell physiology proposed by Hanahan and Weinberg to define cancerous growth (Hanahan and Weinberg 2000) and described below:

Table 2 The role of Hsp70 chaperones in pancreatic cancer pathogenesis
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Self-Sufficiency in Generating Growth Signals in Cancer Cell

Hsp90 is needed to stabilize the fragile structures of many transcription factors and protein kinases that are involved in normal cellular growth pathways. This molecular chaperone is also required to maintain signaling molecules in an active conformation so as to allow rapid triggering by growth signals. In cancer, Hsp90 maintains the activities of the EGFR, HER2 (proto-oncogenic trans-membrane receptor), protein kinases Akt, c-Src and Raf-1(important signaling molecules), CDK4, CDK6 (cell cycle regulator) to promote pancreatic tumour growth, proliferation and survival (Zhang et al. 2008). It also stabilizes the conformations of mutant proteins such as p53, thus allowing these mutated molecules to accumulate within the pancreatic cancer cell (Zhang et al. 2011).

Insensitivity to Anti-growth and Proliferative Signals

Hsp70 has been shown to bind to p53 and other tumor suppressor proteins. Mutation in the p53 protein is one of the most common events in cancer development (Lee et al. 1994). However, although Hsp70 has been shown to accumulate in large amounts in association with mutant p53 protein in cancer cells, there is no conclusive evidence to indicate that increased Hsp levels are necessary to inactivate tumour suppressor molecules for malignant transformation to take place (Lee et al. 1994). On the contrary, Hsp90 plays a very critical role in stabilization of mutant p53 in pancreatic tumor and thereby largely accounts for the enhanced level of oncogenicity (Zhang et al. 2011).

Resistance to Cell Death or Avoidance of Apoptosis

A number of studies showed that elevated levels of Hsp can protect malignant cells against apoptosis generated by therapy (Joly et al. 2010). The apoptotic mechanism follows two pathways, the intrinsic and the extrinsic. For both intrinsic and extrinsic pathways, the ultimate executers of apoptosis are proteases, called caspases, which are activated enzymatically in response to an apoptotic stimulus (Cappello et al. 2002). Due to their cytoprotective role, Hsp have been found to play extremely complex roles in the regulation of apoptosis. They are implicated in both caspase-dependent and independent apoptotic pathways, as well as in the maintenance and activation of anti-apoptotic mediators. Hsp70, which is over expressed in human pancreatic cancer, plays a master regulatory role in inhibition or neutralization of pancreatic cancer associated apoptosis, through imparting TRAI (TNF-related apoptosis-inducing ligand) resistance (Monma et al. 2013), preventing BAX translocation into mitochondria (MacKenzie et al. 2013), inhibiting caspase-9 recruitment and apoptosome formation (Aghdassi et al. 2007) and by providing lysosomal and mitochondrial integrity (Banerjee et al. 2012).

Molecular Chaperones as Anti-cancer Targets in Pancreatic Cancer

There is currently much effort being made to develop more effective Hsp inhibitors for use in pancreatic cancer treatment (Table 3). A large number of chemotherapy combinations has been tested in patients with advanced pancreatic cancer. Only one combination showed significant improvement of survival, however also increased toxicity. Survival of patients with pancreatic cancer could not be improved by the use of these inhibitors. That is why the most important challenge is to define the appropriate way in which to deploy chaperone inhibitors in the treatment of pancreatic cancer.

Table 3 List of potential therapeutic drugs against molecular chaperones in human pancreatic cancer
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Gastric Cancer: An Overview

Gastric Cancer and Hsp70

Among most commonly diagnosed type of cancers in the world Gastric cancer (GC) is the fifth (Bray et al. 2012; IARC (International Agency for Research on Cancer) 2012). Although incidence rates have been declining since the 1990s, significant geographical variations have been observed in the occurrence pattern of GC with some Asian countries and Central and Eastern Europe still showing higher incidence (Bertuccio et al. 2009; Ferro et al. 2014). Anti-sense nucleotides against Hsp70 have been shown to cause cell death in gastric adenocarcinoma cells (Zhao and Shen 2005). In patient samples of gastric tumors, high Hsp 70 levels were associated with poor prognosis (Lee et al. 2013). Reduced levels of Hsp 70 and increased apoptosis were observed in gastric adenocarcinoma cells after triptolide treatment (Arora et al. 2017). On the other hand, Helicobacter pylori, which is among many of those agents that lead to incidences of gastric cancer in human, may bring about a reduced level of Hsp70. Although the detailed pathology of H. pylori induced gastric cancer is not well demonstrated till date, few studies indicate the ability of the expression is one such effect that could be evidenced in patients exhibiting H. pylori infection. A study conducted by Axsen WS and colleagues (Axsen et al. 2009) showed that C57B/L mice infected with human H. pylori undergo reduced expression of not only hsp70 but also hsf1, a transcription factor regulating the expression of hsp70 gene (Axsen et al. 2009). A similar study with human epithelial cell line infected with H. pylori also demonstrated altered proliferation of the cultured cells accompanied by reduced expression of hsp70 (Tao et al. 2014). This altered proliferation is predominantly represented by reduced growth rate. Accordingly, in a separate study, the fate of cultured gastric epithelial cells exposed H. pylori infection was investigated in detail. This study demonstrated increased expression of apoptosis inducing factor and cytosolic cytochrome c leading to increased instances of apoptotic cell death events. Besides, an upregulation of p21 and associated cell cycle modulation could also explain S-phase arrest of the infected cells and subsequently reduced growth rate (Liu et al. 2011). All these studies therefore quite successfully established a negative correlation between H. pylori infection and expression of host hsp70 gene. However, one of the studies led by Ding S. Z. and colleagues provided important molecular insights into the underlying mechanism of H. pylori infection mediated hsp70 down-regulation (Ding et al. 2010). This study showed an epigenetic component in the regulation of hsp70 expression upon H. pylori infection. Phosphorylation on serine 10 of histone H3 in the promoter region of hsp70 gene was shown to condense chromatin thereby inhibiting hsp70 transcription resulting in reduced levels of Hsp70 protein.

Besides altered transcript levels of Hsp70 observed in gastric cancer patients, in many cases differential spatial distribution of the protein within the cell could also be noticed. Like increased translocation of Hsp70 to the nucleus that helps protecting the cell from stress induced nuclear damage. One of the nuclear import carriers of Hsp70 is an evolutionarily conserved protein called Hikeshi (Kose et al. 2012). In a study led by Yanoma T et al., the expression pattern of Hikeshi was investigated in 207 gastric cancer tissue samples. This study showed an elevated expression of this nuclear import protein in gastric cancer tissue samples compared to normal cells. Although apparently this signifies an increased nuclear translocation of Hsp70 as well, no such observation could be noticed at normal temperature. At elevated temperature however, the increased expression of Hikeshi could be well-correlated to increased migration of Hsp70 from cytoplasm to nucleus. Accordingly Hikeshi inhibition in combination with hyperthermia has been suggested as a potential therapeutic tool for refractory gastric cancer (Yanoma et al. 2017). Other therapeutic tools targeting Hsp70 for treatment of gastric cancer includes recently demonstrated cytomegalovirus protein UL138, overexpression of which has been found to induce apoptosis in gastric cancer cell lines (Chen et al. 2016). This is however, in contrary to the common belief that human cytomegalovirus (HCMV) plays an ‘oncomodulatory’ role in neoplastic processes stimulating neoplastic growth (Michaelis et al. 2009).

Hsp70 being an important component of signaling cascades regulating inflammatory responses, therefore constitute a crucial limiting factor affecting successful H. pylori infection. Polymorphisms within the gene sequence controlling the vigor of immune response therefore can be expected to expose individuals to risk of H. pylori infection induced acute gastritis followed by onset of gastric cancer differentially. Oswaldo Partida Rodriguez and colleagues carried out studies in this direction and found out that in a Mexican population a single nucleotide polymorphism in hsp70-1 gene at position +190 is associated with predisposition to gastric cancer and duodenal ulcer (Partida-Rodrıguez et al. 2010). Similarly the BB genotype of hsp70-2 is found to be associated with gastric pre malignant condition in H. pylori infected older patients in a Japanese population (Tahara et al. 2009).

Liver Cancer (Hepatocellular Carcinoma): An Overview

Role of Extracellular Hsp70 in the Progression of Tumor Cell Invasion and Metastasis Leading to Hepatocellular Carcinoma (liver cancer)

Tumor cell invasion and metastasis may be integrated with RhoA. RhoA-mediated regulation of this process takes place through the activation of cytoskeletal proteins that promote myosin interaction with F-actin, leading to contractility. Enhanced contractility of tumor cell greatly aids tumor cell movement and migration ability (Valtcheva et al. 2013). RhoA is an essential member of the rho gene family, which is a homolog of Ras protein (Strutt et al. 1997). Heat shock protein 70 and peptide complexes (eHSP70/HSP70-PCs) regulate Hepatocellualar Carcinoma (HCC) cell migration that occurs via regulation of RhoA expression (Yi et al. 2017).

An array of biological functions of tumor cells is modulated by extracellular heat shock protein 70 and peptide complexes (eHsp70/Hsp70-PCs). The expressions of Hsp70, E-cadherin, α-SMA and phosphorylated-p38 MAPK may characterize malignant potential and could categorize the extent of liver cancer. eHsp70/Hsp70-PCs play a focal role in the EMT of hepatocellular carcinoma through the mediation of the p38/MAPK pathway (Li et al. 2013). EMT is an intricate process that refers to the transmogrification of epithelial cells to stromal cells, where the polarity of epithelial cell fizzles out, consorted by escalated migration and invasion. EMT could be identified by the loss of epithelial cell markers (cadherin and E-cadherin) expression and/or overexpression of mesenchymal cell markers (e.g., α- smooth muscle actin protein, α-SMA) (Gheldof and Berx 2013). The occurrence of liver cancer is closely related to its tumor microenvironment (Gao et al. 2012). A number of components of the tumor microenvironment such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) could be involved with EMT (Jung et al. 2007). Presence of Hsp70 and Hsp70 peptide complexes (Hsp70/Hsp70-PCs) has also been observed in the tumor microenvironment (Schildkopf et al. 2011). Moreover, eHsp70/Hsp70-PCs could also influence a variety of tumor cell functions, such as proliferation and invasion. (Wu et al. 2012; Walsh et al. 2011). A recent study has shown that eHSP70/HSP70-PCs carried out their action through Jun-terminal kinase JNK1/2 signaling pathway in hepatocarcinoma (Zhe et al. 2016). Studies have also demonstrated that the stress-activated mitogen activated protein kinases (MAPK) c-Jun NH2-terminal kinase (JNK) and p38 MAPK greatly influence hepatocarcinogenesis. Both these kinases have important cellular functions and considerable amount of cross-talk between them. Therefore, it is difficult to firmly indicate a specific drug target associated with these pathways (Nakagawa and Maeda 2012).

Role of Hsp70 as DAMP (Damage-Associated Molecular Pattern Molecules): Implications in Liver Cancer (HCC)

In the tumor microenvironment, tumor cells destructed by immune cells release intracellular molecules. Some of these molecules are inflammatory mediators referred to as damage-associated molecular pattern molecules (DAMPs) (Tsai et al. 2014). DAMPs play crucial roles in triggering immune responses and activating repair mechanisms to produce both antitumor and protumor effects. Hsp 70 is a well-characterized DAMP in chronic inflammation (Kang et al. 2013). Stress-inducible Hsp70 functions as a cytoprotective protein when cells are subjected to stressful stimuli. Although, necrotic cells can passively release stress-inducible Hsp70 (Gogate et al. 2012), Hsp70 is also actively released when tumor cells suffer from extrinsic stress. Inside the tumor microenvironment, extracellular Hsp70 can bind to TLR2 and TLR4 expressed by tumor cells and can promote immune tolerance, cancer advancement and promulgation of the tumor microenvironment (Horibe et al. 2014). However, Hsp70 also takes part in averting apoptosis by means of autophagy and can promote a pro-survival mechanism. Eminently, Hsp70 augmented autophagy by means of c-Jun N-terminal kinase (JNK) phosphorylation and Beclin-1 upregulation. Several studies have indicated that inhibition of the autophagy regulator Beclin-1 in tumor cells inhibited stress induced autophagy and high-mobility group protein B1 (HMGB1) release (Schmitt et al. 2007). Numerous tumor cells widely express HMGB1 and upon necrotic cell death it can be secreted or released. Migrating growth cones and malignant cells show high HMGB1 expression. HMGB1 binds tissue-type plasminogen activator and plasminogen, stimulating production of plasmin and tissue invasion (Gong 2013). Secreted HMGB1effectuates responses to infection and injury by achieving high affinity binding with several receptors including receptor for advanced glycation end products (RAGE), TLR2 (Toll-like receptor 2) and TLR4 (Toll-like receptor 4), hence invigorating tumor invasion and metastasis (Chen and Yu 2016). Extracellular Hsp70/Hsp70-PCs can stimulate the HCC cell proliferation through TLR2 and TLR4 activation and subsequent activation of the intracellular JNK1/2/MAPK signaling pathway. Recent studies show, MAPK signaling pathway may mediate signaling through TLRs (Wu et al. 2013). MAPK (p38, ERK1/2, and JNK1/2) signaling pathways can control a number of cellular functions in cancer cells. Previous studies have revealed that cyclin D1 plays a principal role in the normal regulation of the cell cycle (Gong 2013). Cyclin D1 is regulated positively by MAPK, thus exerting influence on tumor cell proliferation (Qiu et al. 2014). Cyclin D1 over-expression could shorten the time required for G1 to S phase transition and elevates cell cycle rate, resulting in rapid and uncontrolled cell proliferation. HCC demonstrates clear evidence of Cell cycle imbalance Overexpression of cyclin D1 has been clearly linked to the occurrence and growth of hepatocarcinoma (Zhang et al. 2015, b). Among different signaling pathways induced by Hsp70, the promotion of tumor growth by activation of the NF-κB cascade has been identified in several tumor cells (Wu et al. 2012). Thus, Hsp70 can enhance both the NF-κB and JNK-Beclin-1/HMGB1 pathways in tumor cells (Zhe et al. 2016). Activation of NF-κB was significant for the effect of Hsp70. H70sp induced a positive feedback loop associating Beclin-1/HMGB1 production, causing re-phosphorylation of NF-κB. This triggered a promotion of cell invasion ability of the cancer cells, a critical aspect of tumor progression (Gong 2013).

Regulatory Effects of Glucose on Heat Shock Response

In the hepatocellular carcinoma cell lines, Glucose can activate HSF1 transcription activity and augment the gene expression of alpha B-crystallin, Hsp70, and oncogenic proteins CSK2, and RBM23 (Mendillo et al. 2012). Glucose can stimulate HSF1 hyper-phoshorylation at serine 326 by activation of the mTOR pathway. The mTOR pathway is upregulated in most tumors and takes part in modulating tumor protein synthesis, cell proliferation, and autophagy by targeting various substances. Inhibition of mTORC1 with rapamycin can significantly suppress the Hsp90 and Hsp70 protein synthesis in breast cancer cell lines (Chou et al. 2012). mTOR C1 kinase can interact with and stimulate the phosphorylation of HSF1/S326 leading to activation of HSF1. Glucose-mTOR is one of the signaling pathways that maintain HSF1 activation in tumor cells. HSF1-mediated heat shock response can in turn regulate the glucose metabolism as well as the proteostasis involved in cell proliferation, metastasis, and therapeutic resistances. The glucose-mTOR-HSF1 pathway is a prospective target for tumor therapy (Ma et al. 2015).

Role of Hsp 70 as Biomarker of Hepatocellular Carcinoma (HCC)

Hepatocellular carcinoma (HCC) is one of the predominant malignancies in the world (Di Tommaso and Roncalli 2017). Many causative factors are connected to HCC occurrence and development, such as chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, consumption of alcohol, and cirrhosis (Gehrmann et al. 2014). It has been observed that tumor suppressor protein (p53) and heat shock protein 70 may exert vital influence in chronic liver diseases. Hsp70 and p53 are frequently observed to be over-expressed in HCC biopsy, especially in advanced HCC (Chuma et al. 2003). They are believed to be putative biomarkers for HCC diagnosis, and proper amalgamation of these 2 markers could improve diagnostic accuracy. It has been reported that both the mRNA and protein levels of Hsp70 increase markedly more in advanced HCC than in early HCC (Di Tommaso et al. 2007). Up-regulation of serum Hsp70 is observed in both liver cirrhotic and HCC patients (Shevtsov and Multhoff 2016).

Conclusions

Over the last few years new vistas leading towards a clearer insight of the molecular basis of GI cancers have opened up. Our knowledge was substantially aided by a better understanding of the genetic basis of the disease and the availability of new information on the role of molecular signaling pathways. Still, many aspects of the functioning of chaperones in disease progression remain unexplored. This review aims to provide a summary of the current knowledge of the role of the Hsp70 and related chaperones in human GI tumorigenesis. Since their initial discovery, significant input has been given towards developing molecular chaperone inhibitors as anticancer agents. The question whether the inhibitors of heat shock proteins would be of any significant help to combat GI cancers still remains to be clearly answered.