Patent Application
20240009174
Prostate cancer (PCa) is the second most diagnosed cancer and third leading cause of cancer related death among American men. First-line treatment regimens for advanced PCa typically target and antagonize the androgen receptor (AR) signaling axis, the main driver of PCa growth. Although current therapies initially alleviate tumor burden, PCa typically recurs with a hormone-resistant phenotype that renders AR-targeting drugs ineffective. Hormone-refractive PCa is treated by taxanes-based chemotherapy, but this is largely palliative. Emergence of neuroendocrine PCa histological variants in some patients relapsing from hormone therapy pose another serious challenge because these cancers are no longer dependent on AR signaling.
Previous studies have demonstrated that mitochondria play important roles in the development of aggressive PCa, which may be attributed to their role in adapting to oxidative stress. Mitochondrial DNA (mtDNA) mutation-induced mitochondrial dysfunction has also been reported in aggressive PCa. To adapt to this pervasive mitochondrial stress, mammalian cells have developed a unique response system known as the mitochondrial unfolded protein response (UPRmt). UPRmt is a stress-induced response, which activates mitochondrial-specific chaperones and proteases to maintain mitochondrial homeostasis. UPRmt is hyperactive and its components are upregulated in various cancers.
There are primarily two major mitochondria specific chaperones identified as heat shock proteins 60 and 10 (HSP60 and HSP10). More than 26 mitochondrial proteases have been identified in mammalian cells with LONP1 and caseinolytic protease proteolytic subunit (ClpP) playing prominent roles. HSP60 is encoded by HSPD1 gene, and is a mitochondrial chaperonin that properly folds nascent or denatured polypeptides. HSP60 monomers self-assemble within the mitochondrion to form a tetradecameric barrel. Damaged or nascent proteins bind to the apical domain of HSP60 within the core of the hydrophobic barrel. The addition of ATP and chaperonin HSP10 induces a charge turnover within the core of the barrel that leads to a conformational shift, which induces protein folding. HSP60 has recently been established as a protein-of-interest in various human diseases including cancer. Increased HSP60 expression predicts poor survival in advanced serous ovarian cancer; inhibits cell death in various tumor cells; is overexpressed in metastatic colon cancer; and is upregulated in gastric cancer cells where it inhibits apoptosis and induces migration. Increased expression of HSP60 correlates with aggressive phenotypes in PCa. These findings establish that HSP60 plays a critical role in maintaining mitochondrial function through the UPRmt. Further, its up regulation in cancer suggests it may facilitate cancer cell growth and survival.
ClpP is a highly conserved mitochondrial serine protease and plays an important role in degradation of unfolded or misfolded proteins. ClpP exists as a heptamer in human mitochondria and relies on AAA+ chaperone ClpX plus ATP to be proteolytically active. ClpP-silencing sensitizes cervical carcinoma cells to the chemotherapeutic agent cisplatin by reducing platinum binding to mtDNA. ClpP is upregulated in primary acute myeloid leukemia (AML) specimens and loss of ClpP significantly decreases viability of AML cells. These findings suggest that both protein folding and protein degradation machinery may be critical in alleviating mitochondrial stress. However, it is not clear whether HSP60:HSP10 folding machinery alone is sufficient or if it requires association with proteases such as ClpP in maintaining UPRmt in cancer.
Based on the foregoing, there is an ongoing and unmet need for additional therapeutic targets beyond the AR signaling axis in order to improve patient outcomes, such as inhibitors of PCa cell growth that are effective on androgen nonresponsive prostate cancer cells.
The present disclosure provides methods and compositions for treating an individual in need of treatment with a compound that inhibits the growth of cancer cells. The methods may be used to disrupt mitochondrial unfolded protein response. In various examples, a compound of this disclosure binds to heat shock protein 60 (HSP60) and inhibits the interaction of HSP60 to ClpP, thus interrupting the mitochondrial unfolded protein response. Without intended to be bound by any particular theory, it is considered targeting HSP60-ClpP mediated UPRmt may provide a novel therapeutic approach for treating advanced prostate cancers that are no longer sensitive to AR targeted therapies.
In an aspect, the present disclosure provides compositions comprising a compound having the following structure:
Without intending to be bound by any particular theory, it is considered DCEM1 binds to the apical domain of HSP60, a region that is critical for interacting with ClpP, and disrupts mitochondrial protein homeostasis leading to cancer cell death.
In an aspect, the present disclosure provides methods of using compositions comprising DCEM1. A method may use a composition of the present disclosure. The compositions of the present disclosure are suitable in methods to treat cancers (e.g., prostate cancer, leukemia, lung cancer, dermatological cancer, premalignant lesions of the upper digestive tract, malignancies of the brain, malignancies of the breast, and the like, and combinations thereof). For example, one or more compositions of the present disclosure can be used to treat cancer and/or induce selective inhibition of the UPRmt. A method of this disclosure may be carried out in combination with one or more known therapy(ies), including, but not limited to, surgery, radiation therapy, chemotherapy, photodynamic therapy, and/or immunotherapy.
For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.
Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. All ranges provided herein include all values that fall within the ranges to the hundredth decimal place, unless indicated otherwise.
The present disclosure provides methods and compositions for treating an individual in need of treatment with a compound that inhibits the growth of cancer cells. The methods may be used to disrupt mitochondrial unfolded protein response. In various examples, a compound of this disclosure binds to heat shock protein 60 (HSP60) and inhibits the interaction of HSP60 to ClpP, thus interrupting the mitochondrial unfolded protein response. Without intended to be bound by any particular theory, it is considered targeting HSP60-ClpP mediated UPRmt may provide a novel therapeutic approach for treating advanced prostate cancers that are no longer sensitive to AR-targeted therapies.
Throughout this application, the singular form encompasses the plural and vice versa. All sections of this application, including any supplementary sections or figures, are fully a part of this application.
The term “treatment” as used herein refers to alleviation of one or more symptoms or features associated with the presence of the particular condition or suspected condition being treated. Treatment does not necessarily mean complete cure or remission, nor does it preclude recurrence or relapses. Treatment can be effected over a short term, over a medium term, or can be a long-term treatment, such as, within the context of a maintenance therapy. Treatment can be continuous or intermittent.
The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The exact amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amount can be determined by one of ordinary skill in the art informed by the instant disclosure using only routine experimentation.
In an aspect, the present disclosure provides compositions comprising a compound having the following structure:
Without intending to be bound by any particular theory, it is considered DCEM1 binds to the apical domain of HSP60, a region that is critical for interacting with ClpP, and disrupts mitochondrial protein homeostasis leading to cancer cell death.
Compositions may comprise DCEM1 and one or more pharmaceutically acceptable carriers. Non-limiting examples of carriers include solutions, suspensions, emulsions, solid injectable compositions that are dissolved or suspended in a solvent before use, and the like. The compositions may be prepared by dissolving, suspending, or emulsifying one or more of the active ingredients in a diluent. Examples of diluents, include, but are not limited to distilled water, physiological saline, vegetable oil, alcohol, dimethyl sulfoxide, and combinations thereof. Further, the compositions may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, etc. The compositions may be sterilized in the final formulation step or prepared by sterile procedure.
The composition of the disclosure may also be formulated into a sterile solid preparation, for example, by freeze-drying, and can be used after sterilized or dissolved in sterile injectable water or other sterile diluent(s) immediately before use. Additional examples of pharmaceutically acceptable carriers include, but are not limited to, sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, including sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations; and combinations thereof. Additional non-limiting examples of pharmaceutically acceptable carriers can be found in: Remington: The Science and Practice of Pharmacy (2012) 22nd Edition, Philadelphia, PA. Lippincott Williams & Wilkins.
In an aspect, the present disclosure provides methods of using compositions comprising DCEM1. A method may use a composition of the present disclosure.
The compositions of the present disclosure are suitable in methods to treat cancers (e.g., prostate cancer, leukemia, lung cancer, dermatological cancer, premalignant lesions of the upper digestive tract, malignancies of the brain, malignancies of the breast, and the like, and combinations thereof). For example, one or more compositions of the present disclosure can be used to treat cancer and/or induce selective inhibition of the UPRmt and/or inhibit the growth/replication of malignant cells and/or metastasis of cancers. A method of this disclosure may be carried out in combination with one or more known therapy(ies), including, but not limited to, surgery, radiation therapy, chemotherapy, photodynamic therapy, and/or immunotherapy.
In various examples, the composition of the present disclosure may be administered in combination with one or more chemotherapy drugs. The composition may be administered sequentially or concurrently with one or more chemotherapy drugs. The sequential administration of the composition and one or more chemotherapy drugs may be separated by seconds, minutes, hours, days, or weeks. Examples of chemotherapy drugs that may be used in combination with the composition include, but are not limited to docetaxel, paclitaxel, enzalutamide, cabazitaxel, abiraterone, etoposide, doxorubicin, tyrosine kinase inhibitors, and the like, and combinations thereof. For example, using a composition comprising DCEM1 in combination with one or more chemotherapy drugs may increase the efficacy of the one or more chemotherapy drugs.
The methods of the present disclosure may be used to inhibit HSP60 from binding ClpP, thus disrupting the UPRmt activity. The UPRmt activity may be altered in vitro or in vivo. Methods of the present disclosure may comprise administering a composition of the present disclosure to an individual exhibiting abnormal HSP60/ClpP expression and/or binding. Examples of conditions in which the present methods and compositions can be used includes, but is not limited to, any condition in which HSP60/ClpP expression and/or binding is abnormal. Such conditions include, but are not limited to, cancer (e.g., prostate cancer and the like), cardiovascular diseases (e.g., coronary artery disease and the like), liver diseases (e.g., fatty liver disease and the like), and neurodegenerative diseases (e.g., Alzheimer's disease, dementia, and the like), and the like. An individual may an individual suspected of or having a condition where HSP60/ClpP expression and/or binding is abnormal.
An individual in need of treatment may have a tumor. The tumor may be from an androgen responsive, androgen non-responsive, and/or a neuroendocrine prostate cancer. A sample of the tumor may be obtained to determine levels of UPRmt and/or HSP60 and/or ClpP. Examples of tumors that can be treated by the present methods or compositions comprising DCEM1 include, but are not limited to, breast adenocarcinoma, glioma, glioblastoma, medulloblastoma, multiple myeloma, melanoma, meningioma, ovarian carcinoma, prostate carcinoma, leukemia, lymphoma, colon carcinoma, pancreatic cancer, hepatic cancer, kidney cancer, sarcoma, and the like, and combinations thereof.
In various examples, the present disclosure provides a method for treating an individual having or suspected of having cancer who has cells (e.g., has a tumor (e.g., tumor sample) having cells) expressing HSP60 and/or ClpP. A sample from the individual may be taken (e.g., tumor biopsy and/or blood sample) and HSP60 and/or ClpP levels may be measured. If the sample (e.g., tumor) is found to have elevated HSP60 and/or ClpP levels, a suitable, therapeutically effective amount of the composition comprising DCEM1 may be administered to the individual using an appropriate route (e.g., intratumoral, intravenous, intradermal injection, oral). The treatment may be carried out without first sampling HSP60 and/or ClpP levels. In various examples, the individual is administered one or more compounds (e.g., DCEM1) that disrupt the UPRmt in cancer cells or a composition comprising one or more compounds that disrupt the UPRmt in cancer cells.
The disclosure provides a method for treating tumors, such as, for example, tumors that comprise cells expressing HSP60 and/or ClpP (e.g., prostate cancer cells). Such tumors may be referred to herein as “HSP60-expressing tumors”, “ClpP-expressing tumors”, and/or “HSP60/ClpP-expressing tumors”. For example, the present disclosure provides a method for reducing the size of a tumor and/or tumor cells; arresting the growth of a tumor and/or tumor cells; and/or reducing the rate of growth of a tumor and/or tumor cells (such as a tumor comprising HSP60/ClpP-expressing cells) or reducing any other symptom that is associated with an individual being afflicted with the tumor, all of which are considered as “treatment” comprising administering to an individual in need of treatment, a therapeutically effective amount of a composition comprising DCEM1, as described herein.
In an example, the current disclosure provides a method of inhibiting growth and/or replication of prostate cancer cells comprising contacting the cells with the small molecule DCEM1 in a therapeutically effective amount and time sufficient to disrupt the UPRmt by inhibiting the interaction of heat-shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) in prostate cancer cells, wherein inhibiting HSP60-ClpP interaction results in inhibition of growth of the prostate cancer cells. In a further example, the prostate cancer cells to be treated can be of a hormone-sensitive, hormone-refractive, and/or neuroendocrine variant.
In an example, the method of the current disclosure provides for the treatment of an individual, comprising:
One or more compositions described herein may be administered to an individual in need of treatment using any known method and route, including oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intracranial injections. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, and subcutaneous administration. Topical and/or transdermal administrations are also encompassed.
In various examples, a subject in need of treatment is administered a therapeutically effective amount of a compound in a composition of the present disclosure. A dose of a therapeutically effective amount of a compound of the present disclosure may have a concentration of 10 nM to 10 mM (e.g., 100 μM), including all 0.1 nM values and ranges therebetween. In an example, a dose of a therapeutically effective amount of a compound in a composition of the present disclosure may have a concentration of 1-500 μM 50-500 μM, 1-250 μM, 10-250 μM, 25-250 μM, 25-150 μM, 50-250 μM, or 50-150 μM.
In an example, an individual in need of treatment is administered a compound or a composition comprising the compound of the present disclosure as a single dose (e.g., a single administration step). Following a single dose, the individual's mitochondrial unfolded protein response activity is decreased for 1-120 hours (hr(s) or h) (e.g., 24-120 hours, 1-48 hours, 12-48 hours, or 24-48 hours), including all second values and ranges therebetween.
In an example, an individual in need of treatment is administered a compound or a composition comprising the compound of the present disclosure in multiple doses dose (e.g., multiple administration steps). Following the multiple doses, the individual's mitochondrial unfolded protein response activity is ameliorated for 1-120 hours (e.g., 24-120 hours, 1-48 hours, 12-48 hours, or 24-48 hours), including all second values and ranges therebetween.
A method may be carried out in a subject in need of treatment who has been diagnosed with or is suspected of having cancer (i.e., therapeutic use). A method can also be carried out in a subject who has a relapse or a high risk of relapse after being treated for cancer.
In an example, a method to inhibit the UPRmt comprises: i) contacting a cancer cell (e.g., a cancer cell in a subject in need of treatment) using DCEM1, where the compound inhibits the interaction of HSP60 and ClpP.
In an example, a compound is used to inhibit the growth and/or replication of cancer cells (e.g., prostate cancer cells). Growth is inhibited by contacting the prostate cancer cells with a compound in an amount (e.g., 1 nM to 1 mM) and time sufficient to inhibit the UPRmt in the cancer cells (e.g., prostate cancer cells), where the inhibition of UPRmt results in inhibition of growth of the cancer cells (e.g., prostate cancer cells). The inhibition of growth may be better than the inhibition of growth caused by other compounds/treatments known in the art. Inhibition of growth refers to any decrease in growth/reproduction of a cell (e.g., the growth/reproduction of cancer cells).
In an example, a method of the present disclosure for treating cancer comprises: i) administering to a subject in need of treatment a composition of the present disclosure, where the compound binds to HSP60 and blocks HSP60 from interacting with ClpP and carrying out the mitochondrial unfolded protein response.
In an example, a method of the present disclosure for reducing the volume of a tumor comprises: i) administering to a subject in need of treatment a composition of the present disclosure, where the compound binds to HSP60 and blocks HSP60 from interacting with ClpP and carrying out the mitochondrial unfolded protein response.
In an example, a method of the present disclosure for preventing cancer metastasis comprises: i) administering to a subject in need of treatment a composition of the present disclosure, where the compound binds to HSP60 and blocks HSP60 from interacting with ClpP, and thus, blocking the mitochondrial unfolded protein response, which may prevent cancer metastasis.
A subject in need of treatment or individual in need of treatment may be a human or non-human mammal. Non-limiting examples of non-human mammals include cows, pigs, mice, rats, rabbits, cats, dogs, or other agricultural animals, pets, service animals, and the like. In various examples, the subject or individual is a male or has male reproductive organs. In various examples, the subject or individual has a prostate.
The steps of the method described in the various examples disclosed herein are sufficient to carry out the methods of the present disclosure. Thus, in an example, the method consists essentially of a combination of the steps of the methods disclosed herein. In another example, a method consists of such steps.
The following Statements provide various examples of the present disclosure:
The following example is presented to illustrate the present disclosure. It is not intended to be limiting in any matter.
The following is an example of use of DCEM1.
Upregulation of UPRmt components associates with poor prognosis in PCa patients. PCa is a disease of aging prostate tissue that exhibits mitochondrial dysfunction, which activates a protective mechanism in mitochondria termed as the UPRmt. To understand the potential impact of UPRmt in PCa, publically available data sets were analyzed to characterize expression of key UPRmt components and to test for correlations with patient outcomes. Transcript levels of HSP60 and ClpP were higher in prostate tumors compared to matched non-tumor prostate tissues (
HSP60 and ClpP silencing inhibit PCa cell survival and proliferation. Activation of UPRmt plays an important role in restoring mitochondrial function and cancer cell survival. Clonogenic assay demonstrated that HSP60 or ClpP-silencing reduced the clonogenicity of several PCa cell lines (
Silencing/knockout of key UPRmt components reduces tumor burden in vivo. To determine the physiological relevance of HSP60 and ClpP on tumor growth, these two genes were silenced in highly invasive androgen-independent PCa cell line PC-3 and performed subcutaneous xenograft studies. Significant reduction in tumor growth was observed upon silencing of HSP60 or ClpP, HSP60-silencing showed higher efficacy in reducing tumor burden (
To test the importance of HSP60 in more physiological PCa models, floxed HSP60 alleles were bred into a genetically engineered mouse model of highly aggressive neuroendocrine PCa in which the Pten, Rb1, and Trp53 tumor suppressor genes were deleted specifically in prostate epithelial cells (TKO). TKO animals have a median survival of 16 weeks. All animals at this age were euthanized as they are expected to have readily detectable prostate tumors. Deletion of both HSP60 alleles significantly decreased prostate tumor weight and volume in TKO animals (
HSP60 regulates expression of ClpP via c-Myc but not vice versa. Decreased ClpP expression was observed in HSP60 KO TKO prostatic tissue (
UPRmt components HSP60 and ClpP localize and interact in mitochondria. Mitochondrial protein folding machinery requires oligomerization of HSP60 and its interaction with HSP10 to maintain mitochondrial proteostasis. In contrast, unfolded proteins are degraded by ClpP proteases to attenuate stress and maintain protein homeostasis under that stress. To understand the functional significance of these mitochondrial proteins, the localization of HSP60 and ClpP in various PCa cells was evaluated. Immunofluorescence and biochemical analysis clearly demonstrated that HSP60 and ClpP co-localize in the mitochondrial matrix (data not shown). Co-immunoprecipitation analysis demonstrated that HSP60 interacts with ClpP in multiple PCa cell lines (
To study the dynamics of HSP60-ClpP interaction, several HSP60 mutants were created and co-transfected with ClpP expressing plasmid in PCa cells. As shown in
Identification of novel inhibitor of HSP60-ClpP interaction that disrupts UPRmt function and induces PCa cell death. Having shown that two arms of UPRmt, HSP60 chaperonin and ClpP protease interact and play critical role in PCa cell survival, an in silico analysis was performed and a small molecule library (developed by Enamine Ltd) was screened to identify drugs targeting the apical domain of HSP60 critical for its interaction with ClpP. 9 compounds (A-I) were identified and screened for their cell death inducing potential in PCa cells (data not shown). It was observed that compound A (referred to as DCEM1) caused robust cell death in PCa cells with no effect on non-carcinoma prostate epithelial RWPE-1 cells (
Since HSP60 chaperonin system is crucial for maintaining mitochondrial homeostasis in normal cells, a novel method, Mitochondrial Chaperonin Activity Assay (MiCAA), was developed to analyze chaperonin activity in live cells using flow cytometry to evaluate whether DCEM1 modulates mitochondrial chaperonin activity. MiCAA demonstrated that DCEM1 did not modulate mitochondrial chaperonin activity in PCa cells (data not shown), whereas ETB and mizoribine, known chaperonin activity inhibitors, significantly inhibited in PCa cells (
Abrogation of HSP60-ClpP interaction by DCEM1 induces metabolic stress in PCa cells. To dissect the underlying mechanism of mitoROS production upon disruption of HSP60-ClpP interaction, OXPHOS Complex I was inhibited using rotenone or OXPHOS Complex III by antimycin-A, the known sources of mitoROS production. It was observed that DCEM1-induced mitoROS production was not attenuated by inhibition of Complex I or III (
Ablation of HSP60-ClpP interaction by DCEM1 downregulates c-Myc, EZH2 and AR as well as inhibits tumor growth in vivo. It was observed that DCEM1 downregulates c-Myc, EZH2, and AR signaling in PCa cells. Decrease in PSA levels also established that DCEM1 disrupt the major drivers of PCa growth and progression (
Discussion. Development of resistance to AR targeted therapy in advanced PCa associates with tumor heterogeneity, which may arise due to varying levels of AR or occurrence of AR variants in prostate tumor cells or emergence of NEPC. The HSP60-ClpP axis was identified as a new target for developing novel PCa therapeutics. The HSP60-ClpP axis is highly upregulated in the majority of prostate tumor tissues, suggesting that this axis provides a novel target for AR-sufficient and AR-deficient/defective prostate tumors. Cancer cells may undergo mitohormesis by constitutively activating cytoprotective mechanisms like UPRmt. It was hypothesized that perturbation of UPRmt would have deleterious effect on growth and progression of PCa. Based on the findings that HSP60 reduces expression of ClpP along with recent findings, inhibiting HSP60 function alone may be sufficient for tumor regression and prevention of recurrence in PCa.
HSP60 and HSP10 are integral components of protein folding machinery in mitochondria, whereas ClpP protease plays a pivotal role in degradation of unfolded proteins. Although other proteases actively coordinates with ClpP proteases in maintaining cellular and mitochondrial proteostasis, only ClpP expression and function were modulated by HSP60 silencing or inhibition. These findings provide evidence that ClpP protease is the key HSP60 client protein from proteolysis machinery to regulate UPRmt in PCa. Drastic decrease in ClpP level and reduced cellular viability upon HSP60-silencing suggests that HSP60 renders its prosurvival function at least in part via ClpP. Increased coexpression of HSP60 and ClpP in PCa cells as well as in clinical PCa specimens suggests that HSP60-ClpP axis is a novel viable target for PCa therapy. The en silico analysis identified several domains of HSP60 and this study showed that HSP60 interacts with ClpP via its apical domain. Deletion of mitochondrial localization signal or inhibition of oligomerization of HSP60 prevent its interaction with ClpP, suggesting that HSP60 interact with ClpP in its oligomeric form via its apical domain in mitochondria.
Although HSP60-silencing renders higher anticancer activities compared to ClpP-silencing, thus providing an opportunity to disrupt the chaperoning activity of HSP60 or HSP60-ClpP interaction for developing potent anticancer agents. HSP60 and ClpP are required for normal and cancer cell survival/function, thus HSP60 knockout or HSP60-silencing or ClpP-silencing is not feasible option in clinics. HSP60 chaperonin activity is also required for normal cellular functioning. Thus, inhibiting HSP60 chaperonin function may have serious deleterious effect that was observed with phase II clinical trials of HSP90 inhibitor on metastatic PCa. This suggest that disrupting HSP60-ClpP interaction may represent a novel and safe target for PCa therapy. Identification of DCEM1 that blocks HSP60-ClpP interaction enhances PCa cell death and inhibit tumor growth in vivo in multiple xenografts and in vivo PCa models represent an unique small molecule for PCa therapy.
A reason for ClpP downregulation is that HSP60-silencing may destabilize ClpP leading to its degradation. Reduced ClpP mRNA upon HSP60-silencing suggests that degradation of ClpP protease is not the sole reason for reduction of ClpP protein. Downregulation of c-Myc by genetic or pharmacological ablation of HSP60 and identification of c-Myc as a transcriptional regulator of ClpP may indicate the involvement of mitochondria-to-nucleus (retrograde) signaling. Thus, it is envisioned that inhibition of HSP60-ClpP interaction by DCEM1 will disrupt mitochondrial homeostasis leading to inhibition of PCa progression and prevention of PCa recurrence.
The majority of mitochondrial proteins are encoded by nuclear DNA and newly synthesized polypeptides are translocated to mitochondria for proper folding. Cancer cells require increased protein synthesis to meet the demands of increased cellular proliferation and accompanying mitochondrial stress, causing activation of UPRmt leading to increased synthesis of HSP60 and ClpP. Increased levels of these two key components of the UPRmt may ultimately influence mitochondrial OXPHOS function leading to increased ROS production, which further enhance UPRmt. Thus there is a continued demand for UPRmt activation in attenuating persistent mitochondrial stresses during PCa tumorigenesis. These findings establish that pharmacological and genetic inhibition of HSP60 and ClpP function decreased the key tumor promoting proteins such as c-Myc and EZH2, ultimately leading to inhibition of cellular proliferation and enhancement of cancer cell death. On the other hand, blocking HSP60-ClpP interaction by DCEM1 will induce metabolic stress causing burst of mitochondrial ROS, shifting balance towards pro-oxidants based therapeutic strategy. Activation of AMPK, inhibition of mTOR pathways, and burst of oxidative stress in response to DCEM1 suggest that abrogation of HSP60-ClpP interaction is a driving force for OXPHOS defects and metabolic stress in PCa.
Although cancer cells prefer aerobic glycolysis for energy, continual production of ATP via OXPHOS system is still required for rapid cancer cell survival and growth. ClpP along with LonP1 degrades Complex I during stress to alleviate mtROS, thus promoting cell survival. HSP60-silencing or inhibition of HSP60-ClpP interaction by DCEM1 contribute to mtROS buildup and mitotoxicity due to inhibition of ClpP function or ClpP-deficiency because ClpP is no longer available/functional in alleviating mtROS production and degrading unfolded protein in mitochondria leading to collapse of mitochondrial function and homeostasis. How HSP60 regulates expression of nuclear-encoded OXPHOS subunits and other mitochondrial machinery is not clearly understood, but this study indicates that HSP60 is a key regulator of mitochondrial-nuclear crosstalk. Mitochondrial proteases such as ClpP trigger mitochondria-to-nuclear signaling pathway and UPRmt activation. Inhibition of ClpP expression and function by HSP60-silencing or HSP60 inhibitor such as DCEM1 may abolish mito-nuclear crosstalk leading to inhibition of retrograde signaling. Inhibition of HSP60 renders mitochondria in a fragile and dysfunctional state leading to enhanced apoptosis and blockage of cellular proliferation. Therefore, chemotherapeutic agents along with HSP60 inhibition may provide a novel combination for therapeutic benefits in PCa. Indeed, it was observed that docetaxel is more efficacious in inducing cell death in HSP60-silenced cells compared to mock cells. Therefore, OXPHOS collapse upon HSP60 and ClpP-silencing or by DCEM1 treatment enhances robust cell death and may be a major reason why cell proliferation and viability is reduced.
Altogether, it was determined that PCa cells display oncogene addiction towards UPRmt components HSP60 and ClpP, as silencing of either proteins inhibit PCa growth and progression. It was also showed that HSP60 and ClpP are interacting proteins and disrupting their interaction with each other had the same effect as silencing. This observation may be of utmost importance as chaperonin activity of HSP60 and protease activity of ClpP are needed by normal cells as well to maintain mitochondrial protein homeostasis but the PLA data described herein showed this interaction is not significant in normal prostate tissue. Therefore, targeting HSP60-ClpP axis in PCa (which is prevalent in PCa regardless of AR status) is a viable option and is expected to hinder PCa progression.
While the present invention has been described through illustrative embodiments, routine modification will be apparent to those skilled in the art and such modifications are intended to be within the scope of this disclosure.
This application claims priority to U.S. Provisional Patent Application No. 63/112,613, filed on Nov. 11, 2020, the disclosure of which is incorporated herein by reference.
This invention was made with government support under grant no. CA160685 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/058863 | 11/10/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63112613 | Nov 2020 | US |
