p62/SQSTM1 indirectly mediates remote multipotent mesenchymal cells and rescues bone loss and bone marrow integrity in ovariectomized rats.

Intramuscular administration of p62/SQSTM1 (sequestosome1)-encoding plasmid demonstrated an anticancer effect in rodent models and dogs as well as a high safety profile and the first evidence of clinical benefits in humans. Also, an anti-inflammatory effect of the plasmid was reported in several rodent disease models. Yet, the mechanisms of action for the p62 plasmid remain unknown. Here, we tested a hypothesis that the p62-plasmid can act through the modulation of bone marrow multipotent mesenchymal cells (MSCs). We demonstrated that a p62 plasmid can affect MSCs indirectly by stimulating p62-transfected cells to secrete an active ingredient(s) sensed by untransfected MSCs. When we transfected MSCs with the p62-plasmid, collected their supernatant, and added it to an untransfected MSCs culture, it switched the differentiation state and prompt osteogenic responses of the untransfected MSCs. According to an accepted viewpoint, ovariectomy leads to bone pathology via dysregulation of MSCs, and restoring the MSC homeostasis would restore ovariectomy-induced bone damage. To validate our in vitro observations in a clinically relevant in vivo model, we administered the p62 plasmid to ovariectomized rats. It partially reversed bone loss and notably reduced adipogenesis with concurrent reestablishing of the MSC subpopulation pool within the bone marrow. Overall, our study suggests that remote modulation of progenitor MSCs via administering a p62-encoding plasmid may constitute a mechanism for its previously reported effects and presents a feasible disease-preventing and/or therapeutic strategy.

The role of Bag3 in cell signaling.

Bag3 has been implicated in a wide variety of physiological processes from autophagy to aggresome formation and from cell transformation to survival. We argue that involvement of Bag3 in many of these processes is due to its distinct function in cell signaling. The structure of Bag3 suggests that it can serve as a scaffold that links molecular chaperones Hsp70 and small Hsps with components of a variety of signaling pathways. Major protein-protein interaction motifs of Bag3 that recruit components of signaling pathways are WW domain and PXXP motif that interacts with SH3-domain proteins. Furthermore, Hsp70-Bag3 appears to be a sensor of abnormal polypeptides during the proteotoxic stress. It also serves as a sensor of a mechanical force during mechanotransduction. Common feature of these and probably certain other sensory mechanisms is that they represent responses to specific kinds of abnormal proteins, i.e. unfolded filamin A in case of mechanotransduction or stalled translating polypeptides in case of sensing proteasome inhibition. Overall Hsp70-Bag3 module represents a novel signaling node that responds to multiple stimuli and controls multiple physiological processes.

P62/SQSTM1 enhances osteogenesis and attenuates inflammatory signals in bone marrow microenvironment.

Bone marrow-derived mesenchymal/stromal stem cells (MSCs) became a major focus of research since the anti-inflammatory features and the osteogenic commitment of these cells can prevent the inflamm-aging and various form of osteopenia in humans and animals. We previously showed that p62/SQSTM1 plasmid can prompt release of anti-inflammatory cytokines/chemokines by MSC when injected in adult mice. Furthermore, it can enhance osteoblastogenesis at the expense of adipogenesis and ameliorate bone density and bone remodeling. On the other hand, absence of p62 partially exhausted MSC pool caused expansion of fat cells within bone marrow and pro-inflammatory mediator's accumulation. Given the critical function of p62 as molecular hub of MSC dynamics, here, using MSCs from p62 knockout adult mice, we investigated the effect of this protein on MSC survival and bone-forming molecule cascades. We found that the main osteogenic routes are impaired in absence of p62. In particular, lack of p62 can suppress Smads activation, and Osterix and CREBs expression, thus significantly modifying the schedule of MSCs differentiation. MSCs obtained from p62-/- mice have also demonstrate an amplified NFκB/ Smad1/5/8 colocalization along with NFκB activation in the nucleus, which precludes Smads binding to target promoters. Considering the "teamwork" of TGFß, PTH and BMP2 on MSC homeostatic behavior, we consider that p62 exerts an essential role as a hub protein. Lastly, ex vivo pulsing p62-deficient MSCs, which then will be administered to a patient as a cell therapy, may be considered as a treatment for bone and bone marrow disorders.

Clinical efficacy of plasmid encoding p62/SQSTM1 (Elenagen) in combination with gemcitabine in patients with platinum-resistant ovarian cancer: a randomized controlled trial.

Background: The purpose of this trial is to evaluate the safety and efficacy of ELENAGEN, a novel anticancer therapeutic DNA plasmid encoding p62/SQSTM1 protein, as an adjuvant to chemotherapy with gemcitabine (GEM) in patients with advanced platinum-resistant ovarian cancer. Methods: This open-label prospective randomized study with two arms. GEM (1000 mg/m2) on days 1 and 8 every 3 weeks was administered in both arms: in the Chemo arm (n = 20), GEM was the only treatment, and in the ELENAGEN arm (n = 20), GEM was supplemented with ELENAGEN (2.5 mg i.m. weekly). The primary endpoint was progression-free survival (PFS), and the secondary endpoint was safety. Antitumor activity was assessed by RECIST 1.1, and criteria safety was assessed according to NCI CTCAE version 5.0. Results: According to the cutoff data, the median follow-up was 13.8 months. There were no serious adverse events related to ELENAGEN treatment. The median PFS was 2.8 and 7.2 months in the Chemo and ELENAGEN arms, respectively (p Log-Rank = 0.03). Notably, at the time of cutoff, 9 patients (45%) in the ELENAGEN arm did not progress, with the longest PFS recorded thus far being 24 months. Subgroup analysis of patients in both arms demonstrated high efficacy of ELENAGEN in patients with worse prognostic factors: high pretreatment levels of CA125 and progression after platinum-free interval <3 months. Conclusions: The addition of ELENAGEN to gemcitabine is effective in patients with platinum-resistant ovarian cancer, including those with a worse prognosis. Clinical trial registration: https://www.clinicaltrials.gov/study/NCT05979298, identifier NCT05979298, 2023-08-07.

P62/SQSTM1 beyond Autophagy: Physiological Role and Therapeutic Applications in Laboratory and Domestic Animals.

Inflammation is the preceding condition for the development of mild and severe pathological conditions, including various forms of osteopenia, cancer, metabolic syndromes, neurological disorders, atherosclerosis, cardiovascular, lung diseases, etc., in human and animals. The inflammatory status is induced by multifarious intracellular signaling cascades, where cytokines, chemokines, arachidonic acid metabolites, adhesion molecules, immune cells and other components foster a "slow burn" at a local or systemic level. Assuming that countering inflammation limits the development of inflammation-based diseases, a series of new side-effects-free therapies was assessed in experimental and domestic animals. Within the targets of the drug candidates for quenching inflammation, an archetypal autophagic gear, the p62/sqstm1 protein, has currently earned attention from researchers. Intracellular p62 has been recently coined as a multi-task tool associated with autophagy, bone remodeling, bone marrow integrity, cancer progression, and the maintenance of systemic homeostasis. Accordingly, p62 can act as an effective suppressor of inflamm-aging, reducing oxidative stress and proinflammatory signals. Such an operational schedule renders this protein an effective watchdog for degenerative diseases and cancer development in laboratory and pet animals. This review summarizes the current findings concerning p62 activities as a molecular hub for cell and tissues metabolism and in a variety of inflammatory diseases and other pathological conditions. It also specifically addresses the applications of exogenous p62 (DNA plasmid) as an anti-inflammatory and homeostatic regulator in the treatment of osteoporosis, metabolic syndrome, age-related macular degeneration and cancer in animals, and the possible application of p62 plasmid in other inflammation-associated diseases.

HSP70s in Breast Cancer: Promoters of Tumorigenesis and Potential Targets/Tools for Therapy.

The high frequency of breast cancer worldwide and the high mortality among women with this malignancy are a serious challenge for modern medicine. A deeper understanding of the mechanisms of carcinogenesis and emergence of metastatic, therapy-resistant breast cancers would help development of novel approaches to better treatment of this disease. The review is dedicated to the role of members of the heat shock protein 70 subfamily (HSP70s or HSPA), mainly inducible HSP70, glucose-regulated protein 78 (GRP78 or HSPA5) and GRP75 (HSPA9 or mortalin), in the development and pathogenesis of breast cancer. Various HSP70-mediated cellular mechanisms and pathways which contribute to the oncogenic transformation of mammary gland epithelium are reviewed, as well as their role in the development of human breast carcinomas with invasive, metastatic traits along with the resistance to host immunity and conventional therapeutics. Additionally, intracellular and cell surface HSP70s are considered as potential targets for therapy or sensitization of breast cancer. We also discuss a clinical implication of Hsp70s and approaches to targeting breast cancer with gene vectors or nanoparticles downregulating HSP70s, natural or synthetic (small molecule) inhibitors of HSP70s, HSP70-binding antibodies, HSP70-derived peptides, and HSP70-based vaccines.

Response of a chemo-resistant triple-negative breast cancer patient to a combination of p62-encoding plasmid, Elenagen, and CMF chemotherapy.

Triple-negative breast cancers are often characterized by aggressive behavior and short clinical course once they become chemotherapy-resistant. We describe below a patient who has shown a response to combination of chemotherapy with Elenagen, a plasmid encoding p62. Elenagen was tested in a previous phase I/II study in patients with refractory solid tumors and shown to be safe. Also, plasmid ability to halt tumor progression and restore sensitivity to chemotherapy was found. Preclinical data supports effects on tumor grade and change the tumor's microenvironment in spontaneous canine breast cancers. We describe here a 48-year old female with triple-negative and BRCA1/2-negative breast cancer who had a primary resistance to chemotherapy and negative dynamics despite the use of multiple lines of treatments. Elenagen was applied intramuscularly at a dose of 1 mg weekly in combination with standard chemotherapy scheme CMF (cyclophosphamide, methotrexate, fluorouracil). In this patient we observed partial tumor regression (by 33%) and 19 weeks of progression-free survival. This first observed objective response to a combination of Elenagen with chemotherapy demonstrates that even in heavily pretreated chemo-resistant triple-negative tumor, the addition of Elenagen to a chemotherapy regimen can cause an objective response and increase in progression-free survival. Such a regimen is worthy of further study in a larger number of patients.

High levels of heat shock protein Hsp72 in cancer cells suppress default senescence pathways.

The major heat shock protein Hsp72 is constitutively expressed in many tumor cell lines and biopsies, and its expression correlates with poor prognosis in several types of cancer. Hsp72 was suggested to play an important role in neoplastic transformation and tumor development. We addressed the role of Hsp72 in cancer cells by investigating the consequences of specific depletion of Hsp72 using small interfering RNA. Down-regulation of Hsp72 in certain cancer lines triggered cell senescence associated with activation and stabilization of p53 and induction of the cell cycle inhibitor p21. Effects of Hsp72 depletion on senescence and p53 did not result from a proteotoxic stress, DNA instability, or activation of ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related pathways. Instead, depletion of Hsp72 reduced stability and activity of the p53 inhibitor Hdm2. In addition, Hsp72 depletion triggered a p53-independent senescence program through inhibitory phosphorylation and down-regulation of the cell cycle kinase Cdc2. Therefore, Hsp72 provides a selective advantage to cancer cells by suppressing default senescence via p53-dependent and p53-independent pathways.

p62-DNA-encoding plasmid reverts tumor grade, changes tumor stroma, and enhances anticancer immunity.

Previously, we reported that the administration of a p62/SQSTM1-encoding plasmid demonstrates high safety and signs of clinical benefits for human cancer patients. The treatment also suppressed tumor growth and metastasis in dogs and mouse models. Here we investigated some mechanistic aspects of these effects. In mammary tumors bearing-dogs, i.m. injections of p62 plasmid reduced tumor sizes and their aggressive potential in 5 out of 6 animals, with one carcinoma switching to adenoma. The treatment increased levels of smooth muscle actin in stroma cells and type III collagen in the extracellular matrix, which correlate with a good clinical prognosis. The p62 treatment also increased the abundance of intratumoral T-cells. Because of the role of adaptive immunity cannot be tested in dogs, we compared the protective effects of the p62 plasmid against B16 melanoma in wild type C57BL/6J mice versus their SCID counterpart lacking lymphocytes. The plasmid was only protective in the wild type strain. Also, p62 plasmid amplified the anti-tumor effect of T-cell transfer from tumor-bearing animals to animals challenged with the same tumors. We conclude that the plasmid acts via re-modeling of the tumor microenvironment, making it more favorable for increased anti-cancer immunity. Thus, the p62-encoding plasmid might be a new adjuvant for cancer treatments.

Targeting heat shock response to sensitize cancer cells to proteasome and Hsp90 inhibitors.

Novel classes of anticancer drugs, including proteasome inhibitors and Hsp90 inhibitors, potently induce heat shock proteins (Hsps). Because Hsps show antiapoptotic activities, we suggested that suppression of such induction may sensitize cancer cells to these drugs. Here, we knocked out the major heat shock transcription factor HSF-1 in several cancer cell lines using small interfering RNA and showed that such cells, which can no longer induce Hsps in response to proteasome and Hsp90 inhibitors, become more sensitive to these drugs. Furthermore, we developed a high-throughput screen for small molecules that inhibit induction of Hsps. The first step was a cell-based screen for inhibitors of Hsps-mediated luciferase refolding followed by a counterscreen for toxicity. The second step was a direct testing for inhibition of Hsp induction by immunoblotting with anti-Hsp72 antibody. After screening of 20,000 compounds from several diversity libraries, we focused on a compound we called NZ28, which potently inhibited induction of Hsps by heat shock, proteasome, and Hsp90 inhibitors in a variety of cell lines, and showed no significant toxicity. After testing of a set of analogues of NZ28, we identified a structural element that was critical for the activity. We also identified another inhibitor of the Hsp induction that was practically nontoxic. This compound, which we called emunin, strongly sensitized myeloma cells to proteasome and Hsp90 inhibitors and prostate carcinoma cells to proteasome inhibitors. This work indicates that targeting the heat shock response may facilitate use of proteasome and Hsp90 inhibitors for cancer treatment.

Cell Death and Survival Assays.

Heat shock proteins are well-known protectors from cell death. Cell death (in particular, apoptosis and necrosis) is accompanied by certain hallmarks manifested as specific alterations in cellular membranes, cytoplasm, nucleus, and mitochondria. Some of those hallmarks are easily detectable in situ and, therefore, they can be applied for the assessment of dying or dead cells. In turn, there are also signs of viable cells that include such features as normal functioning of their membranes and organelles, ability to proliferate, etc. This chapter describes several convenient methods for quantification of dead (apoptotic and necrotic) cells as well as methods for assessment of viable cells. We describe in detail methods of annexin V/propidium iodide (PI) staining, TUNEL assay, Hoechst/PI staining, caspase activation, MTS tetrazolium, lactate dehydrogenase (LDH) release, colony formation, and senescence assays, with the principles, advantages, and drawbacks of each technique.

p62 /SQSTM1 coding plasmid prevents age related macular degeneration in a rat model.

P62/SQSTM1, a multi-domain protein that regulates inflammation, apoptosis, and autophagy, has been linked to age-related pathologies. For example, previously we demonstrated that administration of p62/SQSTM1-encoding plasmid reduced chronic inflammation and alleviated osteoporosis and metabolic syndrome in animal models. Herein, we built upon these findings to investigate effect of the p62-encoding plasmid on an age-related macular degeneration (AMD), a progressive neurodegenerative ocular disease, using spontaneous retinopathy in senescence-accelerated OXYS rats as a model. Overall, the p62DNA decreased the incidence and severity of retinopathy. In retinal pigment epithelium (RPE), p62DNA administration slowed down development of the destructive alterations of RPE cells, including loss of regular hexagonal shape, hypertrophy, and multinucleation. In neuroretina, p62DNA prevented gliosis, retinal thinning, and significantly inhibited microglia/macrophages migration to the outer retina, prohibiting their subretinal accumulation. Taken together, our results suggest that the p62DNA has a strong retinoprotective effect in AMD.

Molecular chaperones regulate p53 and suppress senescence programs.

Many types of cancer cells constitutively express major molecular chaperones at high levels. Recent findings demonstrate that specific depletion of individual chaperones, including various members of the Hsp70 family, small heat shock proteins, or VCP/p97, leads to activation of p53 pathway and subsequently triggers cellular senescence. Here, we discuss a possibility that in cancer cells high levels of chaperones serve to keep the p53 signaling under control, thus allowing cancer cells to evade the default senescence and form tumors.

Multiple thermometers in mammalian cells: why do cells from homeothermic organisms need to measure temperature?

Mammalian cells activate survival signaling pathways and other protective mechanisms or induce apoptotic cell death in response to heat stress at temperatures beyond the range of those that they would ever be expected to encounter in vivo. Recent work has demonstrated that heat shock directly activates the apoptotic proteins Bax and Bak, suggesting that these polypeptides function as cellular thermometers in the mitochondrial apoptotic pathway. Here we review this and other heat-activated signaling pathways and propose a model that postulates that these "cellular thermometers" are not designed to sense physiologically irrelevant temperatures but rather to detect a general buildup of abnormal proteins in the cytosol and other cellular compartments.

Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis.

The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72 Delta EEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72 Delta EEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and Delta Hsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and Delta Hsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.

Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72.

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.

P62 plasmid can alleviate diet-induced obesity and metabolic dysfunctions.

A high-calorie diet (HCD) induces two mutually exacerbating effects contributing to diet-induced obesity (DIO): impaired glucose metabolism and increased food consumption. A link between the metabolic and behavioral manifestations is not well understood yet. We hypothesized that chronic inflammation induced by HCD plays a key role in linking together the two components of diet-induced pathology. Based on this hypothesis, we tested if a plasmid (DNA vaccine) encoding p62 (SQSTM1) would alleviate DIO including its metabolic and/or food consumption abnormalities. Previously we reported that injections of the p62 plasmid reduce chronic inflammation during ovariectomy-induced osteoporosis. Here we found that the p62 plasmid reduced levels of pro-inflammatory cytokines IL-1ß, IL-12, and INFγ and increased levels of anti-inflammatory cytokines IL-4, IL-10 and TGFß in HCD-fed animals. Due to this anti-inflammatory response, we further tested whether the plasmid can alleviate HCD-induced obesity and associated metabolic and feeding impairments. Indeed, p62 plasmid significantly reversed effects of HCD on the body mass index (BMI), levels of glucose, insulin and glycosylated hemoglobin (HbA1c). Furthermore, p62 plasmid partially restored levels of the satiety hormone, serotonin, and tryptophan, simultaneously reducing activity of monoamine oxidase (MAO) in the brain affected by the HCD. Finally, the plasmid partially reversed increased food consumption caused by HCD. Therefore, the administering of p62 plasmid alleviates both metabolic and behavioral components of HCD-induced obesity.

Safety and efficacy of p62 DNA vaccine ELENAGEN in a first-in-human trial in patients with advanced solid tumors.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy.

Increased expression of the major heat shock protein Hsp72 in human prostate carcinoma cells is dispensable for their viability but confers resistance to a variety of anticancer agents.

The major heat shock protein Hsp72 is expressed at high levels in various types of cancer. Here we attempt to clarify the role of Hsp72 in prostate cancer cells by studying the effects of specific downregulation of this protein using siRNA and antisense RNA approaches. Contrary to previous reports, specific depletion of Hsp72 did not reduce viability of the prostate carcinoma cell lines PC-3 and DU-145. However, even short-term downregulation of Hsp72 in these cells made them more sensitive to hyperthermia, inhibitors of proteasome and Hsp90, and tumor necrosis factor. Interestingly, prolonged downregulation of Hsp72 in PC-3 cells over 3 weeks aggravated these effects, as well as enhanced the sensitivity of cells to oxidative stress, radiation, cis-platinum, vinblastin and taxol. The increased sensitivity to the anticancer agents was due to increased apoptosis, as well as other types of cell death, which resulted in the loss of clonogenic survival. Prolonged downregulation of Hsp72 led to severe suppression of the major survival pathways, ERK and NF-kappaB, which may be responsible for enhanced sensitivity of prostate carcinoma cells to a variety of anticancer treatments, as well as reduction of the cell's capability of forming colonies in soft agar.

Anticancer Effects of Targeting Hsp70 in Tumor Stromal Cells.

The stress-induced chaperone protein Hsp70 enables the initiation and progression of many cancers, making it an appealing therapeutic target for development. Here, we show that cancer cells resistant to Hsp70 inhibitors in vitro remain sensitive to them in vivo, revealing the pathogenic significance of Hsp70 in tumor stromal cells rather than tumor cells as widely presumed. Using transgenic mouse models of cancer, we found that expression of Hsp70 in host stromal cells was essential to support tumor growth. Furthermore, genetic ablation or pharmacologic inhibition of Hsp70 suppressed tumor infiltration by macrophages needed to enable tumor growth. Overall, our results illustrate how Hsp70 inhibitors mediate the anticancer effects by targeting both tumor cells and tumor stromal cells, with implications for the broad use of these inhibitors as tools to ablate tumor-associated macrophages that enable malignant progression. Cancer Res; 76(20); 5926-32. ©2016 AACR.