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 transcription-independent mitochondrial cell death pathway is defective in non-transformed cells containing the Pro47Ser variant of p53.

Approximately half of all human cancers contain mutations in the TP53 tumor suppressor. In addition to mutations, there are single nucleotide polymorphisms (SNPs) in TP53 that can dampen p53 function, and can increase cancer risk and decrease the efficacy of cancer therapy. Approximately 6% of Africans and 1% of African-Americans express a p53 allele with a serine instead of proline at position 47 (Pro47Ser, or S47). The S47 variant is associated with increased breast cancer risk in pre-menopausal African Americans, and in a mouse model for the S47 variant, mice are predisposed to spontaneous cancers. We recently showed that the S47 variant is impaired for p53-mediated apoptosis in response to radiation and some genotoxic agents, particularly cisplatin. Here we identify the mechanism for impaired apoptosis of S47 in response to cisplatin. We show that following cisplatin treatment, the S47 variant shows normal stabilization and serine 15 phosphorylation, but reduced ability to bind to the peptidyl prolyl isomerase PIN1, which controls the mitochondrial localization of p53. This is accompanied by impaired mitochondrial localization of S47, along with decreased induction of cleaved caspase-3. Interestingly, we show that this defect occurs only for cisplatin and not for camptothecin. These findings show that normal tissues may respond differently to genotoxic stress depending upon this TP53 genotype. These data suggest that toxicity to cisplatin may be decreased in S47 individuals, and that this compound may be a superior treatment option for these individuals.

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.

ATG5 Mediates a Positive Feedback Loop between Wnt Signaling and Autophagy in Melanoma.

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of ß-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low ß-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased ß-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing ß-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873-85. ©2017 AACR.

Epigenetic remodeling regulates transcriptional changes between ovarian cancer and benign precursors.

Regulation of lineage-restricted transcription factors has been shown to influence malignant transformation in several types of cancer. Whether similar mechanisms are involved in ovarian cancer pathogenesis is unknown. PAX8 is a nuclear transcription factor that controls the embryologic development of the Müllerian system, including the fallopian tubes. Recent studies have shown that fallopian tube secretory epithelial cells (FTSECs) give rise to the most common form of ovarian cancer, high-grade serous ovarian carcinomas (HGSOCs). We designed the present study in order to understand whether changes in gene expression between FTSECs and HGSOCs relate to alterations in PAX8 binding to chromatin. Using whole transcriptome shotgun sequencing (RNA-Seq) after PAX8 knockdown and ChIP-Seq, we show that FTSECs and HGSOCs are distinguished by marked reprogramming of the PAX8 cistrome. Genes that are significantly altered between FTSECs and HGSOCs are enriched near PAX8 binding sites. These sites are also near TEAD binding sites, and these transcriptional changes may be related to PAX8 interactions with the TEAD/YAP1 signaling pathway. These data suggest that transcriptional changes after transformation in ovarian cancer are closely related to epigenetic remodeling in lineage-specific transcription factors.

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.

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.

HSP70 Inhibition Limits FAK-Dependent Invasion and Enhances the Response to Melanoma Treatment with BRAF Inhibitors.

The stress-inducible chaperone protein HSP70 (HSPA1) is implicated in melanoma development, and HSP70 inhibitors exert tumor-specific cytotoxic activity in cancer. In this study, we documented that a significant proportion of melanoma tumors express high levels of HSP70, particularly at advanced stages, and that phospho-FAK (PTK2) and BRAF are HSP70 client proteins. Treatment of melanoma cells with HSP70 inhibitors decreased levels of phospho-FAK along with impaired migration, invasion, and metastasis in vitro and in vivo Moreover, the HSP70 inhibitor PET-16 reduced levels of mutant BRAF, synergized with the BRAF inhibitor PLX4032 in vitro, and enhanced the durability of response to BRAF inhibition in vivo Collectively, these findings provide strong support for HSP70 inhibition as a therapeutic strategy in melanoma, especially as an adjuvant approach for overcoming the resistance to BRAF inhibitors frequently observed in melanoma patients. Cancer Res; 76(9); 2720-30. ©2016 AACR.

Efficacy of the HSP70 inhibitor PET-16 in multiple myeloma.

Multiple myeloma (MM) is a common and largely incurable blood cancer for which new treatment options are needed, as resistance to current modalities is an issue. Additionally, because this tumor type often resides in a hypoxic niche of the bone marrow, new therapeutics that remain effective even under hypoxic conditions are sought. Because of the secretory nature of MM cells they are uniquely under proteotoxic stress, and we hypothesized that these tumor cells may alleviate this stress by upregulating the major stress-induced cytosolic form of the chaperone HSP70. In this work we test the efficacy of the HSP70 inhibitor PET-16 for MM. We show that MM cell lines express significant levels of HSP70, and further that inhibition of HSP70 causes decreased viability and apoptosis, along with proteotoxic stress, as assessed by the accumulation of poly-ubiquitylated proteins. Importantly, we show that growth of these tumor cells under hypoxic conditions has no effect on the ability of PET-16 to be cytotoxic. The HSP70 inhibitor PET-16 should thus be considered further for pre-clinical analyses of efficacy in MM.

Comparison of the activity of three different HSP70 inhibitors on apoptosis, cell cycle arrest, autophagy inhibition, and HSP90 inhibition.

The chaperone HSP70 promotes the survival of cells exposed to many different types of stresses, and is also potently anti-apoptotic. The major stress-induced form of this protein, HSP70-1, is overexpressed in a number of human cancers, yet is negligibly expressed in normal cells. Silencing of the gene encoding HSP70-1 (HSPA1A) is cytotoxic to transformed but not normal cells. Therefore, HSP70 is considered to be a promising cancer drug target, and there has been active interest in the identification and characterization of HSP70 inhibitors for cancer therapy. Because HSP70 behaves in a relatively non-specific manner in the control of protein folding, to date there are no reliably-identified "clients" of this protein, nor is there consensus as to what the phenotypic effects of HSP70 inhibitors are on a cancer cell. Here for the first time we compare three recently-identified HSP70 inhibitors, PES-Cl, MKT-077, and Ver-155008, for their ability to impact some of the known and reported functions of this chaperone; specifically, the ability to inhibit autophagy, to influence the level of HSP90 client proteins, to induce cell cycle arrest, and to inhibit the enzymatic activity of the anaphase-promoting complex/cyclosome (APC/C). We report that all three of these compounds can inhibit autophagy and cause reduced levels of HSP90 client proteins; however, only PES-Cl can inhibit the APC/C and induce G 2/M arrest. Possible reasons for these differences, and the implications for the further development of these prototype compounds as anti-cancer agents, are discussed.

A conserved domain in exon 2 coding for the human and murine ARF tumor suppressor protein is required for autophagy induction.

The ARF tumor suppressor, encoded by the CDKN2A gene, has a well-defined role regulating TP53 stability; this activity maps to exon 1ß of CDKN2A. In contrast, little is known about the function(s) of exon 2 of ARF, which contains the majority of mutations in human cancer. In addition to controlling TP53 stability, ARF also has a role in the induction of autophagy. However, whether the principal molecule involved is full-length ARF, or a small molecular weight variant called smARF, has been controversial. Additionally, whether tumor-derived mutations in exon 2 of CDKN2A affect ARF's autophagy function is unknown. Finally, whereas it is known that silencing or inhibiting TP53 induces autophagy, the contribution of ARF to this induction is unknown. In this report we used multiple autophagy assays to map a region located in the highly conserved 5' end of exon 2 of CDKN2A that is necessary for autophagy induction by both human and murine ARF. We showed that mutations in exon 2 of CDKN2A that affect the coding potential of ARF, but not p16INK4a, all impair the ability of ARF to induce autophagy. We showed that whereas full-length ARF can induce autophagy, our combined data suggest that smARF instead induces mitophagy (selective autophagy of mitochondria), thus potentially resolving some confusion regarding the role of these variants. Finally, we showed that silencing Tp53 induces autophagy in an ARF-dependent manner. Our data indicated that a conserved domain in ARF mediates autophagy, and for the first time they implicate autophagy in ARF's tumor suppressor function.