Harnessing mitochondrial metabolism and drug resistance in non-small cell lung cancer and beyond by blocking heat-shock proteins

for the potential development of new resistance-defying anti-HSP drugs.

Lung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) being the predominant histological subtype.Despite the emergence of targeted and immune-based therapies that have considerably improved the clinical outcomes of selected patients, the overall NSCLC survival rate remains poor.NSCLC patients experience clinical relapse mainly because of chemoresistance.One promising therapeutic approach is targeting specific molecular vulnerabilities that are associated with the metabolic reprogramming of cancer cells.This strategy relies on evidence that cancer cells rewire their metabolism to sustain their uncontrolled growth as well as invasive and metastatic properties, promoting adaptive resistance to chemoradiotherapy.A critical component of this malignant transformation is the increased dependency on high levels of heat shock proteins (HSPs), which support the elevated protein folding demand and quality control of misfolded oncoproteins.Here, we provide an overview of the literature on metabolism reprogramming, deregulation of mitochondrion and on the role of HSPs in promoting malignancy in lung and other cancer types.A particular focus is dedicated to the role of mitochondrial HSP60 (HSPD1) in NSCLC metabolism and drug resistance for the potential development of new resistance-defying anti-HSP drugs.

Lung cancer etiology and pathology
Lung cancer is the second most diagnosed cancer with 2.2 million new cases registered in 2020.It is the leading cause of cancer-related deaths worldwide for both men and women with 1.8 million deaths (Sung et al., 2021).The main risk factor for lung cancer is tobacco smoking accounting for 80-90% of all lung cancer diagnoses, followed by radon exposure (3-14%), and occupational exposure such as asbestos (5-10%) (Schabath and Cote, 2019).Other important risk factors associated with lung cancer are secondhand smoke, cooking fumes, pre-existing non-infectious diseases (e.g., chronic obstructive pulmonary disease) or infectious (e.g., pneumonia) related lung diseases, and inherited genetic susceptibility loci, such as 15q25, 5p15, and 6p21, in non-smokers (McCarthy et al., 2012;Malhotra et al., 2016).
Lung cancer is classified into two main histological groups: small-cell lung (SCLC) and non-small cell lung carcinoma (NSCLC).Non-small cell lung cancer accounts for 85% of all lung cancer cases, including adenocarcinoma (40%), squamous cell carcinoma (20-25%), and large cell carcinoma (10-15%), whereas the remaining 15% are SCLC (Inamura, 2017;Schabath and Cote, 2019).Lung adenocarcinoma (ADC) is defined by the World Health Organization (WHO) as a malignant epithelial neoplasm with glandular differentiation and mucin production or the expression of pneumocyte markers, such as napsin A or TTF-1 (Zheng, 2016).It is located at the periphery of the lung and has a stepwise development defined by the extent of invasiveness, starting with atypical adenomatous hyperplasia, followed by adenocarcinoma in situ.Subsequent stages are characterized by minimally invasive adenocarcinoma, which further evolves into invasive adenocarcinoma (Inamura, 2018).In contrast, squamous cell carcinoma (SqCC) usually arises in the central part of the lung and is defined as a malignant epithelial tumor characterized by keratinization and/or intercellular bridges or expression of squamous cell differentiation markers, such as p40 or p63 (Travis et al., 2015).Large cell lung carcinoma (LCLC) is also located peripherally and is described as an undifferentiated NSCLC that does not display morphological or immunohistochemical features of ADC or SqCC (Travis et al., 2015).In the last few years, different spectra of molecular alterations have been associated with all histological subtypes of lung cancer.Several somatic mutations and chromosomal aberrations that contribute to lung tumorigenesis have been identified (Suster and Mino-Kenudson, 2020).These alterations and mutations affect several oncogenes and tumor suppressor genes, including TP53, KRAS, and LKB1/STK11, which are the three most frequently mutated genes, as well as EGFR, ALK, and HER2.Less frequently, mutations are reported for ROS1, RET, MET, BRAF, NRAS, AKT, MAP2K1, and PIK3CA (Greulich, 2010;Rodriguez-Canales et al., 2016).Moreover, distinct genomic alterations have been found in smokers when compared to non-smokers (Cancer Genome Atlas Research Network, 2014).The identification of such mutations through genetic testing is now used for clinical diagnosis, complemented with standard hematoxylin-eosin staining of the cancerous tissue and the immunohistochemical detection of ADC or SqCC markers (Osmani et al., 2018).

Treatment of NSCLC
Only 15% of patients with newly diagnosed NSCLC are diagnosed with early stage tumors (stages I and II).These patients can benefit from surgical resection, including removal of the so-called lump in combination with adjuvant radiotherapy and chemotherapy, which can delay and sometimes prevent tumor recurrence (Cersosimo, 2002;Vansteenkiste et al., 2014).However, more than half of the patients with NSCLC are diagnosed at a late stage of the disease (stages III and IV), with metastasis representing the major cause of mortality (Osmani et al., 2018).For patients with advanced-stage NSCLC, the standard care has been radiotherapy and platinum-based doublet therapy (e.g., cisplatin combined with another cytotoxic drug; Herbst et al., 2018).However, the identification of specific molecular alterations, discussed above, has enabled the development of molecular targeted therapies.Notably, up to 69% of patients with late-stage NSCLC can potentially benefit from new precision treatments (Hirsch et al., 2017).Patients with EGFR mutations can be treated with first-, second-, or third-generation EGFR tyrosine kinase inhibitors, including erlotinib, afatinib, and osimertinib (Esteban-Villarrubia et al., 2020).Tyrosine kinase inhibitors directed against anaplastic lymphoma kinase protein (ALK), such ascrizotinib and ceritinib, have been proven to be effective in patients with ALK and ROS gene rearrangements (Friboulet et al., 2014).Small molecule inhibitors against the RET, MET, and BRAF kinases improve the clinical outcome of patients in terms of overall and progression-free survival, although the duration of the response is often limited by the insurgence of resistance driven by the occurrence of secondary mutations (Kleczko et al., 2019).In addition to targeted therapies, since 2015, several immunotherapies have been approved for the treatment of advanced NSCLC to disrupt the inhibitory signals that shield cancer cells from immune cell attacks (Ruiz-Cordero and Devine, 2020).Immunotherapies are based on the clinical use of monoclonal antibodies such as ipilimumab, pembrolizumab, and atezolizumab targeting cytotoxic T-lymphocyte antigen 4 (CTLA4), receptor protein programmed cell death protein 1 PD-1, or its ligand PD-L1, respectively (Paz-Ares et al., 2022;Theelen et al., 2021;Broderick, 2020).Several clinical trials have shown a significant improvement in the overall survival of patients; therefore, immunotherapy has now become a first-line treatment for advanced NSCLC, either as monotherapy or in combination with chemotherapy (Arbour and Riely, 2019).Unfortunately, cancer cells can adapt and acquire resistance to immunotherapies by developing defects in interferon-γ signaling or MHC I presentation, which are essential for maintaining potent antitumor responses (Bagchi et al., 2021).Moreover, the enhanced activity of the immune system can lead to the development of immune-related adverse effects, such as pneumonitis or thyroiditis, after treatment of patients with NSCLC with immunotherapies (Johnson et al., 2018).Despite the availability of these new therapies, the 5-year survival rate of patients with advanced-stage lung cancer remains very poor, dropping to 0-10% compared to 60% for early-stage patients, highlighting the need to discover new strategies and therapies for patients who do not benefit from immune checkpoint inhibition (Duma et al., 2019).An emerging and promising approach for the discovery of new molecular targets for anti-cancer therapy is targeting cancer metabolism, and master chaperones, such as heat shock protein (HSP90), have been implicated in cancer metabolic reprogramming (Condelli et al., 2019).In particular, the pharmacologic blockade of the molecular chaperone HSP90 is a promising approach for treating NSCLC tumors characterized by ALK inhibitor resistance (Sang et al., 2013).

Metabolic reprogramming and cancer plasticity
Most malignant cells, among which lung cancer cells are highly proliferative, require the accumulation of nutrients, such as glucose, lipids, amino acids, and nucleotides, to support cell growth and proliferation (Zhu and Thompson, 2019).To sustain these features and survive under stressful conditions, such as hypoxia and nutrient limitation, cancer cells undergo metabolic rewiring or reprogramming (DeBerardinis and Chandel, 2016).Metabolic reprogramming was proposed in 2010 by Hanahan and Weinberg as a new hallmark of cancer, as a consequence of the activation of oncogenes or loss of tumor suppressor activity (Hanahan and Weinberg, 2011).In NSCLC, mutations in genes such as TP53, EGFR, KRAS, LKB1, PI3K, AKT, MYC, and HIF-1 reprogram cell metabolism, resulting in enhanced nutrient uptake and macromolecule biosynthesis, which are necessary to sustain all steps of tumorigenesis (Fig. 1) (Li and Zhang, 2016;Kim and DeBerardinis, 2019).For example, the PI3K/AKT/mTOR pathway promotes the expression of the glucose transporter GLUT1 and the transcription of several glycolytic enzymes, such as hexokinase (HK) and phosphofructokinase-1 (PFK-1), while MYC promotes glutamine metabolism (Vanhove et al., 2019;Pavlova and Thompson, 2016).KRAS stimulates the uptake and utilization of glucose and phospholipids, whereas AKT and downstream mTOR increase protein synthesis (Boroughs and DeBerardinis, 2015;Saxton and Sabatini, 2017).LKB1 (STK11) is a tumor suppressor and upstream regulator of AMP kinase (AMPK), which in turn inhibits fatty acids and protein synthesis (Bonanno et al., 2019).LKB1 deficiency facilitates glycolysis (Zhang et al., 2021).Oncogenic mutations can also occur in metabolic enzymes that generate so-called oncometabolites: 2-hydroxyglutarate (2-HG) is one of these oncometabolites, whose concentration markedly increases in cancerous cells upon mutations in isocitrate dehydrogenase 1or (IDH1/2) (Wang et al., 2020).
Cancer cells reprogram and adapt their metabolism in response to both cell-intrinsic (genetic alterations) and extrinsic signals, which are derived from the extracellular microenvironment comprising soluble (such as oxygen and nutrients) and insoluble factors (such as the extracellular matrix) (Faubert et al., 2020).Moreover, perfusion experiments have shown that these same tumors have areas with various degrees of consequent oxygen and nutrient availability, triggering cells to adapt their metabolism accordingly (Friedl and Alexander, 2011).For example, lung cancer cells rely more on glucose metabolism and oxidative phosphorylation (OXPHOS) in low-perfused areas, whereas in high-perfused areas, they prefer other nutrients, such as fatty acids, amino acids, and lactate, which are then converted to pyruvate and used as a carbon source for the TCA cycle (Hensley et al., 2016).The ability of cells to switch their metabolism is referred to as metabolic adaptability, which is characterized by metabolic flexibility (ability to use different nutrients) and plasticity (ability to use different pathways to metabolize the same fuel) (Fendt et al., 2020).This metabolic plasticity also occurs during cancer progression, supporting the energetic needs of cells through each step (Danhier et al., 2017).For example, it has been shown that during the metastatic process, cancer cells first reduce their mitochondrial respiration to favor detachment from the primary tumor, and then increase it again to migrate and invade (Porporato et al., 2014).The metastasis and chemoresistance inducing process of epithelial to mesenchymal transition (EMT) has also been found regulated by metabolic enzymes in NSCLC and other cancers (Siddiqui et al., 2017;Schwab et al., 2018;Sciacovelli et al., 2016).

Mitochondrial metabolism
Most cancers rely on the functional mitochondria to support carcinogenesis.Oncogenes and tumor suppressors, such as MYC, RAS, mTOR, HIF-1α, and TP53, alter mitochondrial metabolism and function (Fig. 2) (Maycotte et al., 2017).MYC regulates the expression of more than 400 mitochondrial genes, including peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), the main regulator of mitochondrial biogenesis.Gain-of-function mutations in MYC result in increased mitochondrial mass and dependence on OXPHOS (Li et al., 2005).MYC also upregulates mitochondrial function through the induction of glutaminase, which converts glutamine to glutamate and provides a substrate for the TCA cycle (Wise et al., 2008).mTOR can regulate PGC-1α and the expression of some mitochondrial regulators, components of the OXPHOS complexes, and mitochondrial rRNAs, resulting in enhanced mitochondrial biogenesis and oxidative phosphorylation (Morita et al., 2015).PGC-1α is also upregulated by KRAS and AMPK, which can promote mitophagy to eliminate dysfunctional mitochondria and guarantee cell survival under starvation conditions (Vyas et al., 2016).On the other hand, HIF-1α and the inactivation of p53 can inhibit mitochondrial function and OXPHOS, favoring glycolysis through different mechanisms (Liu et al., 2019;Mucaj et al., 2012).
Mitochondrial metabolism can also be affected by direct mutations in nuclear-encoded mitochondrial enzymes, mostly IDH1/2, succinate dehydrogenase (SDH), and fumarate hydratase (FH) (Fig. 2).Mutations in these enzymes lead to the accumulation of the respective oncometabolites (2-HG, succinate, and fumarate), which contribute to cancer transformation.Both succinate and fumarate, intermediates of the TCA cycle, stabilize HIF-1α and switch the cell metabolism from oxidative to glycolytic (Sajnani et al., 2017).Moreover, they can inhibit α-ketoglutarate-dependent enzymes, contributing to cancer transformation with epigenetic alterations (Xiao et al., 2012).2-HG is also associated with chromatin modifications, which are potent inhibitors of α-ketoglutarate-dependent demethylases; however, in contrast to succinate and fumarate, it decreases HIF-1α levels, favoring mitochondrial metabolism (Chowdhury et al., 2011;Koivunen et al., 2012).

Role of metabolism in cancer therapy and drug resistance
The identification of metabolic dependencies and vulnerabilities has led to the development of new small molecules that target key metabolic enzymes and pathways, resulting in tumor growth impairment (Montrose and Galluzzi, 2019).Several approaches are currently being studied, including inhibitors of glycolysis (e.g.,2-deoxy-D-glucose), the TCA cycle, OXPHOS such as complex I inhibitors (e.g., metformin or phenformin), and inhibitors of glutamine and fatty acid metabolism, together with the targeting of nucleotide biosynthesis (Ngoi et al., 2020).Unfortunately, metabolic plasticity and flexibility might limit the efficacy of these anti-metabolic therapies, suggesting the need to either target multiple metabolic pathways simultaneously or target one specific pathway in combination with oncogenic signaling pathways, such as KRAS or MYC, to improve therapy and avoid the insurgence of resistance (DeBerardinis and Chandel, 2016;Méndez-Lucas et al., 2020).Metabolic plasticity has also been shown to contribute to the development of secondary resistance to common anti-cancer therapies.For example, EGFR-mutant NSCLC cells switch from glycolysis to more oxidative metabolism, resulting in EGFR-tyrosine kinase inhibitor resistance (De Rosa et al., 2015).In colorectal cancer, miR-27 alters the oxidative phosphorylation and mitochondria activities contributing to chemoresistance, while HIF-1α-induced glucose metabolic reprogramming is associated to 5-fluorouracil (5-FU) resistance (Barisciano et al., 2020;Dong et al., 2022).It has been shown that small cell lung cancer cells, which undergo metabolic reprogramming relying on the mevalonate-geranylgeranyl diphosphate pathway, are chemoresistant to cisplatin and etoposide (Guo et al., 2022).The ability of cancer cells to reprogram their metabolism as a consequence of the treatment is not the only mechanism involved in the appearance of therapy-resistance; metabolic differences can be found within a tumor (intratumoral heterogeneity) and this can contribute to the selection of more resistant clones (Kreuzaler et al., 2020).For example, it has been observed that melanoma and breast cancer cells with higher oxidative phosphorylation rates tend to be more resistant to reactive oxygen species (ROS)-inducing drugs and neoadjuvant chemotherapy, respectively (Vazquez et al., 2013;Lee et al., 2017).Therefore, cancer metabolism plays a double and opposite role in cancer therapy, as it can be targeted to impair cancer cell growth and promote cell death while contributing to the development of therapeutic resistance, representing a challenge for the effectiveness of combination therapies.

Heat shock proteins
Heat shock proteins (HSPs) are a large family of highly conserved molecular chaperones that are induced in cells in response to environmental and pathological stress conditions such as high temperatures, oxidative stress, hypoxia, drugs, heavy metals, ultraviolet light, inflammation, and infections (Dubrez et al., 2020).Under these stressors, HSPs are indispensable for cell viability and survival, as they preserve and control protein quality, facilitating the folding of newly synthesized proteins or refolding of denatured and misfolded proteins (Bozaykut et al., 2014).Moreover, HSPs are involved in other cellular processes, such as protein assembly, secretion, trafficking, protein degradation, and regulation of transcription factors, thereby maintaining protein homeostasis (Lianos et al., 2015).According to their molecular weights, HSPs are classified into six families: small HSPs, including HSP27 and HSP10, HSP40, HSP60, HSP70, and HSP90 families, and large HSPs, comprising HSP110 and GRP170 (Kampinga et al., 2009).HSP40, also called DNAJ because of the presence of conserved J-domains, is the largest family of HSPs further divided into 3 subclasses: DNAJA, DNAJB, and DNAJC.They interact through their J-domains with HSP70, stimulating its activity, and HSP40 is also called an HSP70 co-chaperone (Faust et al., 2020).The HSP70 family consists of 13 proteins encoded by HSPA genes, and its activity is also regulated by other co-chaperones, such as HSP110, BAG-1, and CHIP (Fernández--Fernández and Valpuesta, 2018).HSPC1-5 genes encode the five members of the HSP90 family, while HSPD and HSPE encode HSP60 and HSP10, respectively.HSP27, HSP70, and HSP90 can directly interact with the surface of unfolded proteins, whereas HSP60 and HSP10 assemble into complexes, generating folding chambers (Calderwood and Gong, 2016).With the exception of small HSPs, which are ATP-independent chaperones, higher molecular HSPs depend on ATP for their activity (Bepperling et al., 2012).Structurally, both HSP70 and HSP90 consist of an N-terminal ATPase domain that uses ATP binding and hydrolysis to allow cycles of protein binding, folding, and release.Small HSPs, instead, lack the ATPase domain and therefore require HSP70 and HSP90 to be released from their 'client' proteins (Haslbeck and Vierling, 2015).The expression of HSPs is regulated by stress-inducible transcription factors, called heat shock factors (HSFs), which transiently bind the heat shock element sequences located upstream of the transcription initiation site of HSP genes (Gomez-Pastor et al., 2018).Under stress conditions, HSF is converted into trimers and translocated from the cytosol into the nucleus, where it binds to the HSP promoters (Puustinen and Sistonen, 2020).Some HSPs (e.g., HSPA8, HSPA9, and HSPA5) are constitutively expressed in cells, and their expression is regulated by physiological conditions, such as cell cycle, differentiation, and apoptosis, subsequently contributing to the regulation of these processes (Jolly and Morimoto, 2000;Hoter et al., 2018).Heat shock protein clients include receptors and signaling kinases, suggesting a central role of HSPs in the regulation of signaling transduction (Streicher, 2019).They are generally localized in the cytosol of cells, but some of them can be found in other cellular compartments such as the nucleus (HSP27), endoplasmic reticulum (GRP78, GRP94, and GRP170), and mitochondria (HSP10, HSP60, DNAJA3, TRAP1, and GRP75) (Chatterjee and Burns, 2017).They can also be secreted into the extracellular milieu either passively from damaged or dead cells or actively via different mechanisms, such as direct translocation, exosomes, vesicles, endosomes, and secretory lysosomes (De Maio and Vazquez, 2013).Once released or localized on the cell surface, extracellular HSPs can interact with extracellular proteins and surface receptors, triggering intracellular signaling via paracrine or autocrine signals (Takeuchi et al., 2015).Both intra-and extracellular HSPs are involved in the development and progression of several diseases, such as neurodegenerative disorders, ischemia, other cardiovascular pathologies, autoimmune diseases, and relevant to this review, cancer (Kaul and Thippeswamy, 2011;Dubey et al., 2015).

Heat shock proteins in cancer
Heat shock proteins are abnormally expressed in several types of cancers, including lung, breast, gastric, colon, prostate, and ovarian cancers, and are often associated with a poor prognosis (Nahleh et al., 2012).Cancer cells require higher metabolic demands and signaling activities than normal cells to sustain their progression and invasion; therefore, they need higher chaperone activities to survive.In fact, the expression levels of oncoproteins are markedly increased in cancerous B. Parma et al. cells, and these proteins are often misfolded; hence, HSPs are required to support the elevated folding demand and to correct misfolded oncoproteins (Chatterjee and Burns, 2017).Heat shock proteins are involved in the regulation of all of the main hallmarks of cancer, including cell proliferation, invasion, metastasis, evasion of apoptosis, and senescence (Fig. 3, Table 1) (Calderwood and Gong, 2016).Many oncoproteins are HSP90 clients, comprising steroid hormone receptors, EGFR, BRAF, IL-6 (JAK/STAT pathway), BCR-ABL, and cyclin-dependent kinase 4 (Cdk4), which contribute to cell proliferation and evasion of anti-growth signals (Schopf et al., 2017).Other clients include p53, apoptotic peptidase activating factor 1 (Apaf-1), AKT, and survivin, which regulate the evasion of apoptosis (Workman et al., 2007).In addition, HSP90 interacts with the human telomerase TERT, limiting senescence, and with hepatocyte growth factor HGF, matrix metallopeptidase MMP2, HIF-1α, and NF-kB, promoting angiogenesis and cell invasion (Kim et al., 2008;Nagaraju et al., 2015;Birbo et al., 2021).Carcinogenesis is also enhanced by HSP70, which has an anti-apoptotic nature that suppresses both the intrinsic and extrinsic apoptotic pathways by blocking Apaf-1 and apoptosis inducing factor AIF-1 and inhibiting the apoptosis regulator BAX and cytochrome c release from the mitochondria, promoting cell death escape (Saleh et al., 2000;Stankiewicz et al., 2005).HSP70 stabilizes p53 and cell cycle protein p21, inducing cell proliferation and inhibiting senescence (Garrido et al., 2006).Moreover, HSP70 promotes autophagy when localized in lysosomes and regulates metastasis through HIF-1α and NF-κB (Daugaard et al., 2007;Colvin et al., 2014).Finally, it can function as a co-chaperone for HSP90 delivering client proteins, such as Cdk4, AKT, and HER2 (Murphy, 2013).Anti-apoptotic effects are also characteristic of HSP27, which regulates p53 signaling and binds to cytochrome c, thereby inhibiting caspase activation (O' Callaghan-Sunol et al., 2007;Bruey et al., 2000).In addition, HSP27 can promote EMT marker expression and cell migration, stimulating metalloproteinase activity and VEGF (Ernst et al., 2020).Pro-survival activity and anti-apoptotic effects are obtained with HSP60, whereas members of the HSP40 family have a dual role in both anti-cancer and pro-cancer processes; for example, DNAJA3, DNAJB4, and DNAJC25 function as tumor suppressors that inhibit cell proliferation, migration, and invasion, while DNAJB1 and DNAJC6 have pro-oncogenic activities (Wu et al., 2017).The promotion of tumor progression and metastasis is also induced by extracellular HSPs, particularly HSP90, HSP70, and HSP27, which can directly interact with extracellular proteins, inducing remodeling of the extracellular matrix and binding to different receptors, such as TLRs, LDL receptor related protein LRP1, EGFR, and CD40, contributing to the activation of signaling pathways that promote EMT, angiogenesis, migration, invasion, and resistance to apoptosis (Seclì et al., 2021).Heat shock proteins represent potential targets for anti-cancer therapy, and different inhibitory approaches are currently being studied, including small-molecule inhibitors, aptamers, antisense oligonucleotides, and antibodies (Chatterjee and Burns, 2017).Moreover, large HSPs are used to develop cancer vaccines by taking advantage of the immunostimulatory properties of HSP110 and GRP170, which can enhance the immunogenicity of protein antigens (Zuo et al., 2016).

Heat shock proteins and metabolism
Heat shock proteins, particularly HSP90 and HSP70, also play vital roles in controlling cancer cell metabolism and metabolic reprogramming.HSP90 interacts with pyruvate kinase PKM2, MYC, and AKT to regulate glycolysis (Yang et al., 2021).Moreover, TRAP1, a mitochondrial member of the HSP90 family, interacts with SDH and regulates mitochondrial respiration via the proto-oncogene tyrosine protein kinase c-Src (Matassa et al., 2018).ATP synthase is one of the TRAP1 interactors, regulating mitochondrial homeostasis and metabolism (Joshi et al., 2020).The effects of regulatory activities mediated by HSPs on cancer metabolism might be different and even opposite depending on cellular context, interacting partners and targets.HSP70 overexpression has also been shown to increase glycolytic metabolism in cancer cells (Wang et al., 2012).The inhibition by the HSP70 inhibitor, pifithrin-µ, of HSPA1A, major member of HSP70 family, reduces the expression of mitochondrial proteins, including components of the electron transport chain, impacting on the mitochondrial respiration (Leu et al., 2017).In breast cancer, GRP75, member of HSP70 family, interacts with estrogen receptor β ensuring a proper mitochondrial transcription and activating the OXPHOS system (Song et al., 2019).In addition, in breast cancer HSP70 increases ROS levels and inhibits

HSP60 (HSPD1) in cancer and drug resistance
HSP60, also called chaperonin or HSPD1, is a nuclear-encoded mitochondrial chaperone essential for mitochondrial protein homeostasis (Ostermann et al., 1989).It is an ATP-dependent chaperone with a cylindrical structure composed of two back-to-back heptameric rings that provide an inner cavity for folding client proteins, thereby avoiding unwanted interactions with other members of the proteome.Following client protein and ATP binding, the cylinder is closed by the two heptameric rings of the co-chaperone HSP10 (Saibil, 2013).Slow ATP hydrolysis allows protein folding through conformational changes, and once hydrolysis is completed, the chamber is re-opened and the protein is released (Koldewey et al., 2017).HSP60 is mainly localized in the mitochondria, where it not only interacts with HSP10 to fold mitochondrial-import proteins, but also with mortalin (HSPA9), survivin, and p53, which regulate apoptosis (Ghosh et al., 2008).HSP60 is also found in the cytosol, where it is implicated in protein trafficking and cellular signaling, promoting pro-survival or pro-apoptotic pathways depending on the situation, or it can be found on the cell surface or released in the extracellular space interacting with the immune system (Cappello et al., 2008).Mutations in HSPD1 have been associated with hereditary neurodegenerative disorders and are involved in autoimmune diseases (Bross and Fernandez-Guerra, 2016;Grundtman and Wick, 2011).It is overexpressed in several liquid and solid tumors, including leukemia, neuroblastoma, breast, colon, lung, gastric, liver, cervical, and prostate cancers, and its increased expression often correlates with poor prognosis (Wu et al., 2017).In cancer cells, mitochondrial HSP60 directly binds to cyclophilin D (CypD), inhibiting CypD-dependent cell death, and interacting with other apoptotic regulators, such as survivin, Bax, p21, and p53, promoting cell survival and cancer progression.Cytosolic HSP60 can also have pro-apoptotic effects promoting caspase activation (Ghosh et al., 2010;Huang and Yeh, 2019).Moreover, it interacts with PI3K, MYC, and β-catenin, favoring oncogenic transformation and tumor metastasis (Tsai et al., 2009;Yan et al., 2015).HSP60 has been shown to mediate drug resistance, and, in ovarian and bladder cancers, HSP60 overexpression correlates with chemoresistance to cisplatin and oxaliplatin, whereas in colorectal cancer, HSP60 inhibition enhances 5-FU sensitivity (Abu-Hadid et al., 1997;Wong et al., 2008).Cisplatin is one of the most common chemotherapeutic drugs used for late-stage lung cancer treatment (Pirker, 2020).It exerts its anti-cancer activity by inducing DNA lesions by interacting with purine bases and stimulating several pathways that lead to apoptosis, but it can also stimulate the production of ROS, triggering cell death (Dasari and Tchounwou, 2014;Ghosh, 2019).One of the main reasons for chemotherapy failure in NSCLC is the development of cisplatin resistance (Ashrafizadeh et al., 2021).In Parma et al. (2021) NSCLC cells were treated with low doses of cisplatin and HSP60-targeting molecule to avoid affecting cell proliferation or survival.When combined, cisplatin and the HSP60-targeting molecule appeared to act synergistically by increasing ROS production.Therefore, HSP60 chemical inhibition increases cisplatin sensitivity in NSCLC cells, requiring a lower dose of cisplatin to slow down or kill the cancerous cells, potentially breaking cisplatin resistance.To date, only a few small molecules have been developed to target HSP60, including some natural and synthetic compounds (Meng et al., 2018).The antibiotics mizoribine, myrtucommulone A, and synthetic o-carboranylphenoxyacetanilide are known to inhibit the folding activity of HSP60 (Ban et al., 2010;Tanabe et al., 2012;Wiechmann et al., 2017).In particular, mizoribine has been found to induce the differentiation of non-lymphocytic leukemia cells, while myrtucommulone A induced apoptosis in several cancer cells and abrogated the EMT of bladder cancer cells (Inai et al., 1997;Izgi et al., 2015;Iskender et al., 2016).Geldanamycin, an antibiotic known to bind to and inhibit HSP90, has been associated with HSP60 loss in osteosarcoma cells (Gorska et al., 2013).Sinularin can decrease HSP60 expression in melanoma cells, and the proteasome inhibitor bortezomib has been reported to hyperactivate HSP60 expression on the cell surface of ovarian cancer, leading to phagocytosis (Su et al., 2012;Chang et al., 2012).Finally, Polson et al. (2018) identified the synthetic small molecule KHS101 as an HSP60-targeting compound, which interferes with the mitochondrial metabolism of glioblastoma cells, strikingly suppressing cell growth in vitro and in vivo and promoting cell death.In particular, KHS101 induces the formation of irreversible multi-aggregates of HSP60 with metabolic enzymes involved in glycolysis, the TCA cycle, OXPHOS, and mitochondrial integrity (Polson et al., 2018).Other studies have shown that HSP60 is fundamental for ensuring proper mitochondrial proteostasis and oxidative metabolism and supporting cell growth and progression both in vitro and in vivo in several cancers, including pancreatic, ovarian, and kidney cancers (Zhou et al., 2018;Guo et al., 2019;Teng et al., 2019).

HSP60 and NSCLC
HSP60 has been found overexpressed in NSCLC, correlated with poor prognosis (Parma et al., 2021;Tang et al., 2021) and it has been proposed as a disease biomarker (Xu et al., 2011;Fucarino and Pitruzzella, 2020).However, little is known about its biological role in NSCLC, with some contradictory observations.For instance, it has been demonstrated that HSP60 favors the mitochondrial importation of the FHIT protein, contributing indirectly to trigger the apoptotic process (Druck et al., 2019), but also that it can bind to pro-caspase 3 to oppose apoptosis or to p53 to prevent senescence (Campanella et al., 2008;Marino Gammazza et al., 2017).More recently, HSP60 has been demonstrated to be an essential gene for NSCLC proliferation and survival, and a strong metabolic breakdown has been observed upon its genetic loss or via the KHS101 inhibitor, making it an interesting therapeutic target (Parma et al., 2021) (Fig. 4).In line with these data, a fitness analysis on previously published pan-cancer and genome-wide CRISPR/Cas9 cellular screenings revealed the high dependency of both ADC and SqCC tumors on the HSP60 gene, confirming its fundamental importance for the disease (Parma et al., 2021).Importantly, by different types of bioinformatic analyses and genetic screenings, it was found that no single genetic alteration can protect NSCLC cells from the lethal effects of KHS101 (Parma et al., 2021), suggesting a potentially broad clinical application, with the possibility of avoiding the selective pressure to form resistant cells and the consequent insurgence of drug resistance.It will be therefore important to continue the preclinical investigations and better elucidate the HSP60 dependency of NSCLCs to further explore the emerging therapeutic opportunities.

Conclusions
Heat shock proteins are attractive targets for anti-cancer therapy, and several efforts have been made to develop inhibitors targeting the main HSPs.However, few studies have focused on the targeting of HSP60, whose role in sustaining mitochondrial metabolism makes it a good candidate for anti-cancer therapy.Recent studies demonstrate the effective suppression of cell proliferation and promotion of cell death of several cancer cells following HSP60 knockdown or chemical B. Parma et al. interference with the small molecule KHS101 (Polson et al., 2018;Zhou et al., 2018;Guo et al., 2019;Parma et al., 2021).As a consequence of HSP60 loss, cancer cells undergo strong mitochondrial metabolic impairment, making HSP60 a targetable metabolic vulnerability in cancer cells and enhancing chemotherapy drug sensitivity (Wong et al., 2008;Parma et al., 2021).Future studies should be conducted to test other known HSP60 modulators on NSCLC cells to evaluate their potential efficiency in suppressing cell growth and mitochondrial metabolism in vitro and in vivo, or to develop new drugs with higher affinities for HSP60, taking advantage of rapidly emerging targeting techniques based on the induction of selective protein degradation (Khan et al., 2020;Burslem and Crews, 2020).Fig. 4. Metabolic alterations upon HSP60 loss.HSP60 (HSPD1) interacts with and folds mitochondrial-imported proteins, including metabolic enzymes, regulating mitochondrial metabolism, and apoptotic proteins.The lack of HSP60 impairs mitochondrial metabolism and ATP synthesis, resulting in cell growth arrest, cell death, and increased sensitivity to chemotherapeutic drugs.ROS, reactive oxygen species; OXPHOS, oxidative phosphorylation.

Table 1
Summary of known targets of HSPs in cancer cells.

Table 2
Summary of known HSP inhibitors in cancer.