The Identification of CELSR3 and Other Potential Cell Surface Targets in Neuroendocrine Prostate Cancer.

Although recent efforts have led to the development of highly effective androgen receptor (AR)-directed therapies for the treatment of advanced prostate cancer, a significant subset of patients will progress with resistant disease including AR-negative tumors that display neuroendocrine features [neuroendocrine prostate cancer (NEPC)]. On the basis of RNA sequencing (RNA-seq) data from a clinical cohort of tissue from benign prostate, locally advanced prostate cancer, metastatic castration-resistant prostate cancer and NEPC, we developed a multi-step bioinformatics pipeline to identify NEPC-specific, overexpressed gene transcripts that encode cell surface proteins. This included the identification of known NEPC surface protein CEACAM5 as well as other potentially targetable proteins (e.g., HMMR and CESLR3). We further showed that cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) knockdown results in reduced NEPC tumor cell proliferation and migration in vitro. We provide in vivo data including laser capture microdissection followed by RNA-seq data supporting a causal role of CELSR3 in the development and/or maintenance of the phenotype associated with NEPC. Finally, we provide initial data that suggests CELSR3 is a target for T-cell redirection therapeutics. Further work is now needed to fully evaluate the utility of targeting CELSR3 with T-cell redirection or other similar therapeutics as a potential new strategy for patients with NEPC. Significance: The development of effective treatment for patients with NEPC remains an unmet clinical need. We have identified specific surface proteins, including CELSR3, that may serve as novel biomarkers or therapeutic targets for NEPC.

Increased mTOR activity and metabolic efficiency in mouse and human cells containing the African-centric tumor-predisposing p53 variant Pro47Ser.

The Pro47Ser variant of p53 (S47) exists in African-descent populations and is associated with increased cancer risk in humans and mice. Due to impaired repression of the cystine importer Slc7a11, S47 cells show increased glutathione (GSH) accumulation compared to cells with wild -type p53. We show that mice containing the S47 variant display increased mTOR activity and oxidative metabolism, as well as larger size, improved metabolic efficiency, and signs of superior fitness. Mechanistically, we show that mTOR and its positive regulator Rheb display increased association in S47 cells; this is due to an altered redox state of GAPDH in S47 cells that inhibits its ability to bind and sequester Rheb. Compounds that decrease glutathione normalize GAPDH-Rheb complexes and mTOR activity in S47 cells. This study reveals a novel layer of regulation of mTOR by p53, and raises the possibility that this variant may have been selected for in early Africa.

A Novel Inhibitor of HSP70 Induces Mitochondrial Toxicity and Immune Cell Recruitment in Tumors.

The protein chaperone HSP70 is overexpressed in many cancers including colorectal cancer, where overexpression is associated with poor survival. We report here the creation of a uniquely acting HSP70 inhibitor (HSP70i) that targets multiple compartments in the cancer cell, including mitochondria. This inhibitor was mitochondria toxic and cytotoxic to colorectal cancer cells, but not to normal colon epithelial cells. Inhibition of HSP70 was efficacious as a single agent in primary and metastatic models of colorectal cancer and enabled identification of novel mitochondrial client proteins for HSP70. In a syngeneic colorectal cancer model, the inhibitor increased immune cell recruitment into tumors. Cells treated with the inhibitor secreted danger-associated molecular patterns (DAMP), including ATP and HMGB1, and functioned effectively as a tumor vaccine. Interestingly, the unique properties of this HSP70i in the disruption of mitochondrial function and the inhibition of proteostasis both contributed to DAMP release. This HSP70i constitutes a promising therapeutic opportunity in colorectal cancer and may exhibit antitumor activity against other tumor types. SIGNIFICANCE: These findings describe a novel HSP70i that disrupts mitochondrial proteostasis, demonstrating single-agent efficacy that induces immunogenic cell death in treated tumors.

Paradoxical Role for Wild-Type p53 in Driving Therapy Resistance in Melanoma.

Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.

Fatty acid transport protein 2 reprograms neutrophils in cancer.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that are crucial for the regulation of immune responses in cancer. These cells contribute to the failure of cancer therapies and are associated with poor clinical outcomes. Despite recent advances in the understanding of PMN-MDSC biology, the mechanisms responsible for the pathological activation of neutrophils are not well defined, and this limits the selective targeting of these cells. Here we report that mouse and human PMN-MDSCs exclusively upregulate fatty acid transport protein 2 (FATP2). Overexpression of FATP2 in PMN-MDSCs was controlled by granulocyte-macrophage colony-stimulating factor, through the activation of the STAT5 transcription factor. Deletion of FATP2 abrogated the suppressive activity of PMN-MDSCs. The main mechanism of FATP2-mediated suppressive activity involved the uptake of arachidonic acid and the synthesis of prostaglandin E2. The selective pharmacological inhibition of FATP2 abrogated the activity of PMN-MDSCs and substantially delayed tumour progression. In combination with checkpoint inhibitors, FATP2 inhibition blocked tumour progression in mice. Thus, FATP2 mediates the acquisition of immunosuppressive activity by PMN-MDSCs and represents a target to inhibit the functions of PMN-MDSCs selectively and to improve the efficiency of cancer therapy.

Tailoring Chemotherapy for the African-Centric S47 Variant of TP53.

The tumor suppressor TP53 is the most frequently mutated gene in human cancer and serves to restrict tumor initiation and progression. Single-nucleotide polymorphisms (SNP) in TP53 and p53 pathway genes can have a marked impact on p53 tumor suppressor function, and some have been associated with increased cancer risk and impaired response to therapy. Approximately 6% of Africans and 1% of African Americans express a p53 allele with a serine instead of proline at position 47 (Pro47Ser). This SNP impairs p53-mediated apoptosis in response to radiation and genotoxic agents and is associated with increased cancer risk in humans and in a mouse model. In this study, we compared the ability of wild-type (WT) and S47 p53 to suppress tumor development and respond to therapy. Our goal was to find therapeutic compounds that are more, not less, efficacious in S47 tumors. We identified the superior efficacy of two agents, cisplatin and BET inhibitors, on S47 tumors compared with WT. Cisplatin caused dramatic decreases in the progression of S47 tumors by activating the p53/PIN1 axis to drive the mitochondrial cell death program. These findings serve as important proof of principle that chemotherapy can be tailored to p53 genotype.Significance: A rare African-derived radioresistant p53 SNP provides proof of principle that chemotherapy can be tailored to TP53 genotype. Cancer Res; 78(19); 5694-705. ©2018 AACR.

The p53 Tumor Suppressor in the Control of Metabolism and Ferroptosis.

The p53 tumor suppressor continues to be distinguished as the most frequently mutated gene in human cancer. It is widely believed that the ability of p53 to induce senescence and programmed cell death underlies the tumor suppressor functions of p53. However, p53 has a number of other functions that recent data strongly implicate in tumor suppression, particularly with regard to the control of metabolism and ferroptosis (iron- and lipid-peroxide-mediated cell death) by p53. As reviewed here, the roles of p53 in the control of metabolism and ferroptosis are complex. Wild-type (WT) p53 negatively regulates lipid synthesis and glycolysis in normal and tumor cells, and positively regulates oxidative phosphorylation and lipid catabolism. Mutant p53 in tumor cells does the converse, positively regulating lipid synthesis and glycolysis. The role of p53 in ferroptosis is even more complex: in normal tissues, WT p53 appears to positively regulate ferroptosis, and this pathway appears to play a role in the ability of basal, unstressed p53 to suppress tumor initiation and development. In tumors, other regulators of ferroptosis supersede p53's role, and WT p53 appears to play a limited role; instead, mutant p53 sensitizes tumor cells to ferroptosis. By clearly elucidating the roles of WT and mutant p53 in metabolism and ferroptosis, and establishing these roles in tumor suppression, emerging research promises to yield new therapeutic avenues for cancer and metabolic diseases.

Mutant p53 controls tumor metabolism and metastasis by regulating PGC-1α.

Mutant forms of p53 protein often possess protumorigenic functions, conferring increased survival and migration to tumor cells via their "gain-of-function" activity. Whether and how a common polymorphism in TP53 at amino acid 72 (Pro72Arg; referred to here as P72 and R72) impacts this gain of function has not been determined. We show that mutant p53 enhances migration and metastasis of tumors through the ability to bind and regulate PGC-1α and that this regulation is markedly impacted by the codon 72 polymorphism. Tumor cells with the R72 variant of mutant p53 show increased PGC-1α function along with greatly increased mitochondrial function and metastatic capability. Breast cancers containing mutant p53 and the R72 variant show poorer prognosis compared with P72. The combined results reveal PGC-1α as a novel "gain-of-function" partner of mutant p53 and indicate that the codon 72 polymorphism influences the impact of mutant p53 on metabolism and metastasis.

PUMA-dependent apoptosis in NSCLC cancer cells by a dimeric ß-carboline.

Dimeric ß-carbolines are cytotoxic against multiple NSCLC cell lines, and we report herein our preliminary studies on the mechanism of action of these dimeric structures. Dimeric ß-carboline 1, which is more potent than the corresponding monomer in NSCLC cell lines, is a lysosomotropic agent that inhibits autophagy and mediates cell death by apoptosis, upregulating the pro-apoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis) in a dose dependent manner.

A link between TP53 polymorphisms and metabolism.

Besides being a critical tumor suppressor, the TP53 gene also plays a role in metabolism and recent studies in humans have implicated the codon 72 polymorphism of TP53 in this role. Using a humanized knock-in mouse model for these TP53 variants, we show that this polymorphism has a significant impact on the metabolic response to a high-fat diet.

The African-specific S47 polymorphism of p53 alters chemosensitivity.

The TP53 protein is known to affect the sensitivity of tumor cells to cell death by DNA damaging agents. We recently reported that human and mouse cells containing an African-specific coding region variant of p53, Pro47Ser (hereafter S47), are impaired in the transactivation of a small subset of p53 target genes including GLS2 and SCO2, and are markedly resistant to cisplatin. Further, mice containing this variant are markedly predisposed to cancer. Together these findings suggested that cancer-affected humans with the S47 variant might not be effectively treated with cisplatin. To more directly test this premise, we created transformed derivatives of mouse embryo fibroblasts (MEFs) containing wild type p53 (WT) and the S47 variant and analyzed them for chemosensitivity. We find that transformation with E1A and Ras actually reverses the chemosensitivity/transcriptional differences between WT p53 and S47. Specifically, E1A/Ras-transformed S47 cells show increased sensitivity to cisplatin and paclitaxel, and comparable transactivation of GLS2 and SCO2, compared to cells with WT p53. These data suggest that the functional differences between WT p53 and S47 in primary cells may not hold true for transformed cells. They also offer hope that cisplatin and paclitaxel may be effective chemotherapeutic drugs for S47 individuals with cancer.

Genetic Modifiers of the p53 Pathway.

The tumor suppressor gene TP53 is the most frequently mutated gene in human cancer; this gene is subject to inactivation by mutation or deletion in >50% of sporadic cancers. Genes that encode proteins that regulate p53 function, such as MDM2, MDM4, and CDKN2A (p14(ARF)) are also frequently altered in tumors, and it is generally believed that the p53 pathway is likely to be inactivated by mutation in close to 100% of human tumors. Unlike most other cancer-relevant signaling pathways, some of the genes in the p53 pathway contain functionally significant single nucleotide polymorphisms (SNPs) that alter the amplitude of signaling by this protein. These variants, thus, have the potential to impact cancer risk, progression, and the efficacy of radiation and chemotherapy. In addition, the p53 pathway plays a role in other biological processes, including metabolism and reproductive fitness, so these variants have the potential to modify other diseases as well. Here we have chosen five polymorphisms in three genes in the p53 pathway for review, two in TP53, two in MDM2, and one in MDM4. These five variants were selected based on the quality and reproducibility of functional data associated with them, as well as the convincingness of epidemiological data in support of their association with disease. We also highlight two other polymorphisms that may affect p53 signaling, but for which functional or association data are still forthcoming (KITLG and ANRIL). Finally, we touch on three questions regarding genetic modifiers of the p53 pathway: Why did these variants arise? Were they under selection pressure? And, is there compelling evidence to support genotyping these variants to better predict disease risk and prognosis?

An African-specific polymorphism in the TP53 gene impairs p53 tumor suppressor function in a mouse model.

A nonsynonymous single-nucleotide polymorphism at codon 47 in TP53 exists in African-descent populations (P47S, rs1800371; referred to here as S47). Here we report that, in human cell lines and a mouse model, the S47 variant exhibits a modest decrease in apoptosis in response to most genotoxic stresses compared with wild-type p53 but exhibits a significant defect in cell death induced by cisplatin. We show that, compared with wild-type p53, S47 has nearly indistinguishable transcriptional function but shows impaired ability to transactivate a subset of p53 target genes, including two involved in metabolism:Gls2(glutaminase 2) and Sco2 We also show that human and mouse cells expressing the S47 variant are markedly resistant to cell death by agents that induce ferroptosis (iron-mediated nonapoptotic cell death). We show that mice expressing S47 in homozygous or heterozygous form are susceptible to spontaneous cancers of diverse histological types. Our data suggest that the S47 variant may contribute to increased cancer risk in individuals of African descent, and our findings highlight the need to assess the contribution of this variant to cancer risk in these populations. These data also confirm the potential relevance of metabolism and ferroptosis to tumor suppression by p53.

The P72R Polymorphism of p53 Predisposes to Obesity and Metabolic Dysfunction.

p53 is well known for its tumor suppressor role, but this protein also has a poorly understood role in the regulation of metabolism. Human studies have implicated a common polymorphism at codon 72 of p53 in diabetic and pre-diabetic phenotypes. To understand this role, we utilized a humanized mouse model of the p53 codon 72 variants and monitored these mice following challenge with a high-fat diet (HFD). Mice with the arginine 72 (R72) variant of p53 developed more-severe obesity and glucose intolerance on a HFD, compared to mice with the proline 72 variant (P72). R72 mice developed insulin resistance, islet hypertrophy, increased infiltration of immune cells, and fatty liver disease. Gene expression analyses and studies with small-molecule inhibitors indicate that the p53 target genes Tnf and Npc1l1 underlie this phenotype. These results shed light on the role of p53 in obesity, metabolism, and inflammation.

Design, Synthesis and Biological Evaluation of ß-Carboline Dimers Based on the Structure of Neokauluamine.

The design, synthesis and biological evaluation (anticancer and antimalarial activity) of bis-ß-carbolines, based on the structure of the naturally occurring alkaloid neokauluamine, is described.

Differentiation state-specific mitochondrial dynamic regulatory networks are revealed by global transcriptional analysis of the developing chicken lens.

The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that requires a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected more than 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, more than 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic (DUT, PDK1, SNPH), autophagy (ATG3, ATG4B, BECN1, FYCO1, WIPI1), and mitophagy (BNIP3L/NIX, BNIP3, PARK2, p62/SQSTM1) transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells.

Suppression of MAPK/JNK-MTORC1 signaling leads to premature loss of organelles and nuclei by autophagy during terminal differentiation of lens fiber cells.

Although autophagic pathways are essential to developmental processes, many questions still remain regarding the initiation signals that regulate autophagy in the context of differentiation. To address these questions we studied the ocular lens, as the programmed elimination of nuclei and organelles occurs in a precisely regulated spatiotemporal manner to form the organelle-free zone (OFZ), a characteristic essential for vision acuity. Here, we report our discovery that inactivation of MAPK/JNK induces autophagy for formation of the OFZ through its regulation of MTORC1, where MAPK/JNK signaling is required for both MTOR activation and RPTOR/RAPTOR phosphorylation. Autophagy pathway proteins including ULK1, BECN1/Beclin 1, and MAP1LC3B2/LC3B-II were upregulated in the presence of inhibitors to either MAPK/JNK or MTOR, inducing autophagic loss of organelles to form the OFZ. These results reveal that MAPK/JNK is a positive regulator of MTORC1 signaling and its developmentally regulated inactivation provides an inducing signal for the coordinated autophagic removal of nuclei and organelles required for lens function.

Endogenous hydrogen peroxide production in the epithelium of the developing embryonic lens.

PURPOSE: Hydrogen peroxide (H2O2) is an endogenously produced reactive oxygen species (ROS) present in a variety of mammalian systems. This particular ROS can play dichotomous roles, being beneficial in some cases and deleterious in others, which reflects the level and location of H2O2 production. While much is known about the redox regulation of ROS by antioxidant and repair systems in the lens, little is known about the endogenous production of H2O2 in embryonic lens tissue or the physiologic relevance of endogenous H2O2 to lens development. This gap in knowledge exists primarily from a lack of reagents that can specifically detect endogenous H2O2 in the intact lens. Here, using a recently developed chemoselective fluorescent boronate probe, peroxyfluor-6 acetoxymethyl ester (PF6-AM), which selectively detects H2O2 over related ROS, we examined the endogenous H2O2 signals in the embryonic lens. METHODS: Embryonic day 10 chick whole lenses in ex vivo organ culture and lens epithelial cells in primary culture were loaded with the H2O2 probe PF6-AM. To determine the relationship between localization of mitochondria with active membrane potential and the region of H2O2 production in the lens, cells were exposed to the mitochondrial probe MitoTracker Red CMXRos together with PF6-AM. Diphenyleneiodonium (DPI), a flavin inhibitor that blocks generation of intracellular ROS production, was used to confirm that the signal from PF6-AM was due to endogenous ROS production. All imaging was performed by live confocal microscopy. RESULTS: PF6-AM detected endogenous H2O2 in lens epithelial cells in whole lenses in ex vivo culture and in lens epithelial cells grown in primary culture. No endogenous H2O2 signal could be detected in differentiating lens fiber cells with this probe. Treatment with DPI markedly attenuated the fluorescence signal from the peroxide-specific probe PF6-AM in the lens epithelium, suggesting that basal generation of ROS occurs in this region. The lens epithelial cells producing an endogenous H2O2 signal were also rich in actively respiring mitochondria. CONCLUSIONS: PF6-AM can be used as an effective reagent to detect the presence and localization of endogenous H2O2 in live lens cells.