Introduction enhancing the cell survival. Aside from

Introduction

The
Hsp70 family, consisting of multiple ATP-dependent molecular chaperone proteins
of ~70kDa molecular mass, plays a major role in maintaining protein homeostasis
among both prokaryotes and eukaryotes by mediating the correct protein
folding.  The importance of these
proteins is reflected by their abundance in most living organisms and strong
homology among the family members, rarely ranging below 50% amino-acid sequence
identity.

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The
major role of the Hsp70 proteins is maintaining the proteins homeostasis under
heat-shock conditions, though the Hsp70-related refolding process is also enhanced
by various stress-inducing stimuli (such as oxidative stress, hypoxia and
altered pH), since the general cellular stress induces misfolding and aggregation
of proteins, resulting in interference upon regulatory complexes. Upon stress
conditions, the Hsp70 proteins act as a robust defense system, unfolding the
misfolded or aggregated proteins and keeping them in a folding-competent state,
enhancing the cell survival. Aside from the main role in maintaining proteostasis,
eukaryotic Hsp70s also assist in signaling and protein trafficking, affecting
various cell survival pathways.

Structure
and mechanism of action of Hsp70 proteins

The
highly-conserved domain structure of Hsp70 proteins consists of N-terminal
nucleotide-binding ATPase domain (NBD), a flexible interdomain linker region
and C-terminal substrate-binding domain (SBD), containing a
substrate-stabilizing “lid (Bertelsen et al., 2009). Due to their low intrinsic
ATP hydrolysis rate (Russell et al., 1998), Hsp70s closely cooperate with various
co-chaperones, which stimulate their ATPase activity and support substrate binding.
Precisely, the Hsp40 co-chaperones (belonging to the J-protein superfamily) in
complex with a protein substrate bind the NBD of Hsp70, inducing ATP hydrolysis
and subsequent large-scale conformational shift, which results in stabilizing
the Hsp70-substrate complex and dissociation of Hsp40. Nucleotide-exchange
factors (NEFs), such as E. coli GrpE,
catalyze the release of ADP, allowing the binding of ATP molecule, which leads
to release of substrate from the Hsp70 under a reverting conformational change
(Kityk et al., 2012, Fan et al., 2003)(Fig.1 from Kumar, et al., 2016). Another
class of co-chaperones bind to the C-terminal domain of Hsp70, coupling its
function with HSP90 activity and ubiquitin-mediated degratadion (Allan et al.,
2011).

            A
distinctive function of Hsp70s, not present in Hsp90 protein family, is the lack
of their specific client protein, as they bind to the exposed hydrophobic
patches of misfolded and unfolded proteins. Interestingly, Hsp70 is rather an
unfoldase than a refoldase, increasing the available pool of unfolded proteins,
diminishing the aggregation and allowing the return to the native state
(Kellner et al. 2014).

Hsp70 expression
in H. sapiens

The
human HSP70 family consists of eight proteins, despite few exceptions, having
overlapping function in the cell (Daugaard et al., 2007), and majority of them
is constitutively expressed. An important exception is HSP70-1 protein, its expression
controlled, among other factors, by Sp1, c-MYC and Foxa1 (Morgan, 1989),
causing it to be preferentially expressed in the G1 and S phases of
cell cycle (Milarski et al., 1986, Taira et al., 1997). Moreover, HSP70-1
expression is rapidly induced under stress conditions, controlled by the
heat-shock inducible transcription factor HSF1, which under normal conditions
is kept in an inactive complex with cytosolic HSP70 and HSP90 (Akerfelt et al.,
2010). Accumulation of misfolded proteins in the cell redirects HSP90 from
HSF1, allowing it to bind HSP70-1 promoter and induce its transcription.

Hsp70s
and apoptosis

            Apoptosis is the central anti-cancer
defense mechanism in organism, multiple apoptosis pathways induced by various anti-cancer
drugs. Highly expressed Hsp70s interfere at several points of these pathways:
activation of caspases, release of cytochrome c and generation of reactive
oxygen species (Mosser et al. 2007). It is known that inhibition of Hsp70
increases cell sensitivity to apoptosis (Nylansted et al. 2000). Hsp70 inhibits
the intrinsic apoptotic pathway by interacting with nerve growth factor and
subsequent activation of the PI3K/Akt survival pathway, also stabilizing the
Akt-PKB complex. Moreover, Hsp70s inhibit the stress-activated  kinase ASK-1, blocks C-Jun N-terminal kinase
JNK, dampening JNK-mediated apoptosis (Gabai et al. 2000). Hsp70s also affect
the expression of Bcl2-associated transcription factors, inhibit the formation
of apoptosome by interaction with its ATPase domain, regulate the folding and
function of the caspase-activated DNase CAD and restore DNA integrity by
interaction with PARP1 formation of the protein repair complex (Kumar et al.
2016). In addition, Hsp70 blocks the extrinsic pathway by reducing the
caspase-8/10-mediated cleavage of Bid (Matsumori et al. 2006) and counteracts
the caspase-independent apoptotic pathway by blocking the AIF-induced chromatin
condensation (Ravagnan et al. 2001). Overall, Hsp70s exhibit potent
antiapoptotic activity.

Hsp70s
and cancer

In
view of its importance to cell survival and stress response, it is not
surprising that HSP70-1 is frequently overexpressed in cells that underwent
malignant transformation. The factor that gives rise to HSP70-1 overexpression
is the high level of proteotoxic stress in tumors, activating HSF1, however at
least three cancer-relevant activators of HSF1 have been discovered, leading to
common overexpression of Hsp70 gene in cancer cells: the longevity factor
SIRT1, p53 family member ?Np63? and mTORC1 (Murphy, 2013). Moreover, high Hsp70
levels indicate a tumorgenic, chemotherapy resistant phenotype (Rerole et al.
2011), due to the Hsp70 cytoprotective effect, predicting a poor prognosis in
endometrial cancers, renal cell tumors and osteosarcomas (Ciocca, Calderwood
2005). Hsp70s are also overexpressed in early stages of prostate cancer and
imatinib-resistant chronic myeloid leukemia (overexpression of Hsp70 helps
resist imatinib-mediated cell death) (Kumar et al. 2016). In addition, HSP70-2
is overexpressed during progression of breast cancer. Overexpression of Hsp70
is also related to poor prognoses and resistance to chemotherapy and radiotherapy
in breast cancer, gastric cancer and acute leukemia (Kumar et al. 2016). These
facts, together with ability of Hsp70 to inhibit multiple pathways of
apoptosis, make Hsp70 inhibitors a promising therapeutic agent, possibly
impacting anti-cancer drug resistance.

The
possibility of sensitizing or killing tumor cells by Hsp70 inhibition becomes
even more promising, considering the fact that down-regulation of Hsp70, having
no effect on regular cells, is cytotoxic to transformed cells (Schmitt et al.
2006), meaning that constitutively stressed tumor cells are dependent on the
protective functions of Hsp70s.

Targeting Hsp70 in cancer treatment

            Both Hsp70 and Hsp90 function is
strictly dependent on nucleotide-mediated regulation, making them a druggable
target for cancer treatment by inhibiting Hsp70 ATPase activity. In contrary to
Hsp90, few inhibitors are known for Hsp70s. One of the possible mechanisms of
Hsp70 regulation include HSF1 inhibition to block Hsp70 expression. Inhibition
of HSF1-mediated Hsp70 expression can be achieved by use of quercetin,
diterpenetriperoxide and triptolide, along with benzopyrene. This approach is
supposed not to affect other HSP proteins (Kumar et al. 2016). On the other
hand, blocking Hsp70-AIF interaction with AIF-derived peptides also show
promise – ADD70 (AIF-derived decoy for Hsp70) was shown to decrease tumor sizes
in rat melanomas and colon cancers, additionally sensitizing tumors to
cisplatin and increasing tumor infiltration by CD8+ T-cells (Schmitt
et al 2006). Another approach is the application 2-phenylacetylene sulfonamide
(PES) as a C-terminal domain inhibitor and expression repressor, possibly
inducing the caspase-dependent apoptotic pathway (Steele et al. 2009).

It
has been shown that targeting the N-terminal ATP-ase domain of Hsp70 with a
adenosine-derived VEK-155008 blocks the ATPase activity and induces death of
carcinoma cells (Massey et al, 2010). Unfortunately, animal trials of the
compound are yet to be conducted. Other compounds targeting the N-terminal
domain, such as azure C, methylene blue and myricetin, strongly inhibit Hsp70
activity, their specificity, however, is still unknown. Surprisingly, MKT-077 –
a cationic rhodacyanine dye that acts as a mitochondrial Hsp70 N-terminal
domain inhibitor, despite not interacting directly with Hsp70, is currently
being tested in Phase I clinical trials (Koya et al. 1996). NSC 630668 and
MAL3-101 both inhibit the ATP-ase activity and proliferation of SK-BK-3 cancer
cells (Evans et al. 2006).

            Distrupting the interaction between
Hsp70 and its co-chaperones is another possible therapeutic strategy, because
of the strong dependence of Hsp70 activity on co-chaperone presence.
Pyrimidotriazinediones, a new class of drugs that interacts with