The transcription factor sex-determining region Y-box 2 (SOX2) in bladder cancer.

Sex-determining region Y-box 2 (SOX2) is a transcription factor with a central role in embryologic development. SOX2 is also an oncogene in several cancer types. Prior work by our group has shown SOX2 activity associates with cell cycle dysregulation in early-stage bladder cancer. The present study was thus undertaken to broadly investigate SOX2 in bladder cancer, with emphasis on associations with tumor stage, clinical outcomes, and tumorigenicity. Gene expression was quantified by immunohistochemistry in an established tissue microarray (n=303 cystectomy specimens, all stages) and whole tissue sections of noninvasive papillary urothelial carcinoma (n=25). Gene expression by RNA sequencing was evaluated in non-muscle invasive and muscle-invasive cohorts from publicly available repositories. By immunohistochemistry, SOX2 was expressed in 40% of whole tissue sections of noninvasive papillary carcinoma, which correlated with SOX2 expression by RNA sequencing (r=0.6, P=0.001, Spearman correlation). Expression tended to be focal (median H-score =6). SOX2 was expressed in only 9% of TMA cases, consistent with focal expression. SOX2 expression was substantially higher in muscle-invasive compared with noninvasive papillary urothelial carcinoma by RNA sequencing (P<0.001, Wilcoxon rank sum test). SOX2 expression associated with stage progression in lamina-propria invasive cancers (hazard ratio =2, P=0.05, Cox model, binary, RNA sequencing) but not noninvasive papillary cancers (P=0.5, Cox model, binary, RNA sequencing). SOX2 expression did not associate with overall survival in muscle-invasive carcinoma. Activity of SOX2 in bladder cancer was tested in vivo using murine allografts created with MB49 cells that express human SOX2 (MB49-SOX). MB49-SOX allografts expressed this protein focally by immunohistochemistry, much like human tumors. Compared with controls, MB49 allografts demonstrated larger tumor size (P=0.03, Wilcoxon rank sum test) and higher tumor burden in mesenteric metastases (P=0.009, Wilcoxon rank sum test). Though SOX2 expression is focal within tumors, it may drive tumorigenesis, increase growth rate, and promote aggressive features of bladder cancer, particularly stage progression of early-stage disease.

International Society of Urological Pathology Consensus Conference on Current Issues in Bladder Cancer. Working Group 4: Molecular Subtypes of Bladder Cancer-Principles of Classification and Emerging Clinical Utility.

Molecular subtyping has been a major focus of bladder cancer research over the past decade. Despite many promising associations with clinical outcomes and treatment response, its clinical impact has yet to be defined. As part of the 2022 International Society of Urological Pathology Conference on Bladder Cancer, we reviewed the current state of the science for bladder cancer molecular subtyping. Our review included several different subtyping systems. We derived the following 7 principles, which summarize progress and challenges of molecular subtyping: (1) bladder cancer has 3 major molecular subtypes: luminal, basal-squamous, and neuroendocrine; (2) signatures of the tumor microenvironment differ greatly among bladder cancers, particularly among luminal tumors; (3) luminal bladder cancers are biologically diverse, and much of this diversity results from differences in features unrelated to the tumor microenvironment, such as FGFR3 signaling and RB1 inactivation; (4) molecular subtype of bladder cancer associates with tumor stage and histomorphology; (5) many subtyping systems include idiosyncrasies, such as subtypes recognized by no other system; (6) there are broad fuzzy borders between molecular subtypes, and cases that fall on these fuzzy borders are often classified differently by different subtyping systems; and (7) when there are histomorphologically distinct regions within a single tumor, the molecular subtypes of these regions are often discordant. We reviewed several use cases for molecular subtyping, highlighting their promise as clinical biomarkers. Finally, we conclude that data are currently insufficient to support the routine use of molecular subtyping to guide bladder cancer management, an opinion shared with the majority of conference attendees. We also conclude that molecular subtype should not be considered an "intrinsic" property of a tumor but should instead be considered the result of a specific laboratory test, performed using a specific testing platform and classification algorithm, validated for a specific clinical application.

Urothelium-Specific Expression of Mutationally Activated Pik3ca Initiates Early Lesions of Noninvasive Bladder Cancer.

Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an in vivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3caH1047R mouse model expressing one or two R26-Pik3caH1047R alleles in a urothelium-specific manner was generated. Pik3caH1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3caH1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3caH1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3caH1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations.

Clinical and pathological characteristics of metastatic tumors to the urinary bladder.

Metastatic carcinoma to the urinary bladder is a rare and under-recognized condition in surgical pathology. In this study, we identified 8 cases of true bladder metastasis at our institution in the past 20 years, excluding secondary tumors via direct extension or serosal implantation. The most common tumor type is malignant melanoma (3/8), followed by clear cell renal cell carcinoma (2/8), adenocarcinoma of the gastrointestinal tract (2/8), and breast invasive lobular carcinoma (1/8). There are 6 cases of endoscopically exophytic metastasis and 2 cases of diffuse metastasis, commensurate with 6 patients with hematuria and 2 patients with urinary obstruction as respective clinical symptoms. Exophytic bladder metastasis usually presents with similar clinical features as urothelial carcinoma, while diffuse metastasis often masquerades as a urinary tract infection. In the latter circumstance, a markedly thickened bladder wall discerned via imaging study is the best indication for the bladder biopsy to circumvent the misdiagnosis. Histologically, the metastatic tumors can also mimic conventional urothelial carcinoma or its histological variants, and thus pose a potential diagnostic challenge to pathologists. The lack of an in situ component in primary bladder cancer may hint at bladder metastasis. Accurate diagnosis of bladder metastasis requires heightened alertness to this rare condition in addition to a multidisciplinary approach.

Histologic Analysis of Buccal Graft Quality Stratified by Tobacco Use in Patients Undergoing Substitution Urethroplasty.

OBJECTIVE: To determine if there are histologic differences relative to tobacco exposure in buccal mucosa. Substitution urethroplasty outcomes may be worse in tobacco users and we investigate if the buccal graft is inherently damaged due to chronic tobacco exposure. METHODS: Subjects undergoing substitution urethroplasty with buccal graft harvest were prospectively consented in this IRB approved study. Subjects with poor dentition were excluded. A detailed tobacco use history was obtained. Cotinine testing was performed day of surgery to confirm or exclude active tobacco use. Trimmed portions of harvested graft were sent for tissue processing. Standard hematoxylin and eosin staining was performed. A single blinded pathologist performed analysis of the slides. Using a scale of none, mild, moderate, or severe slides were analyzed for cytologic atypia, architectural complexity, inflammation, and keratinization. Evidence of vascular damage was noted and the type of inflammation if present was classified. RESULTS: Twenty-five buccal grafts were analyzed. No evidence of vascular damage or cytologic atypia were noted in any grafts. While mild architectural complexity and mild inflammation, typically lymphocytic, were noted in several of the buccal mucosa sections, this did not appear to correlate with tobacco exposure. The majority of grafts demonstrating increased keratinization correlated with significant tobacco exposure, but this was not consistently noted in all those with tobacco use. CONCLUSIONS: Buccal mucosa in patients with tobacco exposure did not show significant histologic alterations. Outcomes of substitution urethroplasty may be more impacted by persistent systemic exposure causing local ischemia as opposed to the graft tissue itself.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

A transcriptional network of cell cycle dysregulation in noninvasive papillary urothelial carcinoma.

Human cancers display a restricted set of expression profiles, despite diverse mutational drivers. This has led to the hypothesis that select sets of transcription factors act on similar target genes as an integrated network, buffering a tumor's transcriptional state. Noninvasive papillary urothelial carcinoma (NIPUC) with higher cell cycle activity has higher risk of recurrence and progression. In this paper, we describe a transcriptional network of cell cycle dysregulation in NIPUC, which was delineated using the ARACNe algorithm applied to expression data from a new cohort (n = 81, RNA sequencing), and two previously published cohorts. The transcriptional network comprised 121 transcription factors, including the pluripotency factors SOX2 and SALL4, the sex hormone binding receptors ESR1 and PGR, and multiple homeobox factors. Of these 121 transcription factors, 65 and 56 were more active in tumors with greater and less cell cycle activity, respectively. When clustered by activity of these transcription factors, tumors divided into High Cell Cycle versus Low Cell Cycle groups. Tumors in the High Cell Cycle group demonstrated greater mutational burden and copy number instability. A putative mutational driver of cell cycle dysregulation, such as homozygous loss of CDKN2A, was found in only 50% of High Cell Cycle NIPUC, suggesting a prominent role of transcription factor activity in driving cell cycle dysregulation. Activity of the 121 transcription factors strongly associated with expression of EZH2 and other members of the PRC2 complex, suggesting regulation by this complex influences expression of the transcription factors in this network. Activity of transcription factors in this network also associated with signatures of pluripotency and epithelial-to-mesenchymal transition (EMT), suggesting they play a role in driving evolution to invasive carcinoma. Consistent with this, these transcription factors differed in activity between NIPUC and invasive urothelial carcinoma.

Intratumoral Heterogeneity Promotes Collective Cancer Invasion through NOTCH1 Variation.

Cellular and molecular heterogeneity within tumors has long been associated with the progression of cancer to an aggressive phenotype and a poor prognosis. However, how such intratumoral heterogeneity contributes to the invasiveness of cancer is largely unknown. Here, using a tumor bioengineering approach, we investigate the interaction between molecular subtypes within bladder microtumors and the corresponding effects on their invasiveness. Our results reveal heterogeneous microtumors formed by multiple molecular subtypes possess enhanced invasiveness compared to individual cells, even when both cells are not invasive individually. To examine the molecular mechanism of intratumoral heterogeneity mediated invasiveness, live single cell biosensing, RNA interference, and CRISPR-Cas9 gene editing approaches were applied to investigate and control the composition of the microtumors. An agent-based computational model was also developed to evaluate the influence of NOTCH1 variation on DLL4 expression within a microtumor. The data indicate that intratumoral variation in NOTCH1 expression can lead to upregulation of DLL4 expression within the microtumor and enhancement of microtumor invasiveness. Overall, our results reveal a novel mechanism of heterogeneity mediated invasiveness through intratumoral variation of gene expression.

An integrated multi-omics analysis identifies prognostic molecular subtypes of non-muscle-invasive bladder cancer.

The molecular landscape in non-muscle-invasive bladder cancer (NMIBC) is characterized by large biological heterogeneity with variable clinical outcomes. Here, we perform an integrative multi-omics analysis of patients diagnosed with NMIBC (n = 834). Transcriptomic analysis identifies four classes (1, 2a, 2b and 3) reflecting tumor biology and disease aggressiveness. Both transcriptome-based subtyping and the level of chromosomal instability provide independent prognostic value beyond established prognostic clinicopathological parameters. High chromosomal instability, p53-pathway disruption and APOBEC-related mutations are significantly associated with transcriptomic class 2a and poor outcome. RNA-derived immune cell infiltration is associated with chromosomally unstable tumors and enriched in class 2b. Spatial proteomics analysis confirms the higher infiltration of class 2b tumors and demonstrates an association between higher immune cell infiltration and lower recurrence rates. Finally, the independent prognostic value of the transcriptomic classes is documented in 1228 validation samples using a single sample classification tool. The classifier provides a framework for biomarker discovery and for optimizing treatment and surveillance in next-generation clinical trials.

[Molecular pathology of urogenital tumors : Recommendations from the 2019 International Society of Urological Pathology (ISUP) Consensus Conference]. / Molekularpathologie bei urologischen Tumoren : Empfehlungen der Konsenskonferenz der Internationalen Gesellschaft für Uropathologie (ISUP) 2019.

Comprehensive understanding of molecular principles in cancer and the diversification of oncological therapy promise individual therapeutic concepts, which have not yet found their way into urogenital cancer therapy. In March 2019 the International Society of Urogenital Pathology (ISUP) therefore held a consensus conference on recommendations for molecular diagnostics of genitourinary tumors, which were published in five separate manuscripts and are summarized in this article.In preparation for the conference, a comprehensive survey of current practices for molecular testing of urogenital tumors was carried out by members of the ISUP. At the conference, the results and the corresponding background information were presented by five working groups and recommendations for action for diagnostics were developed. An agreement between 66% of the conference participants was defined as consensus.

Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

Mutational profile of endometrial hyperplasia and risk of progression to endometrioid adenocarcinoma.

BACKGROUND: Endometrial hyperplasia is a precursor to endometrioid adenocarcinoma (EMC), the most common uterine cancer. The likelihood of progression to carcinoma may be evaluated by histologic subclassification of endometrial hyperplasia, although these subclasses are subjective and only modestly reproducible among pathologists. Patient care would be improved by a more objective test to predict the risk of cancer progression. METHODS: Next-generation sequencing was performed on archived endometrial biopsy specimens from a retrospective cohort of women with endometrial hyperplasia. Cases were considered to be either progressing if the patient subsequently developed EMC or resolving if the patient had a subsequent negative tissue sampling or no cancer during medium-term follow-up (32 patients: 15 progressing and 17 resolving). Somatic mutations in endometrial hyperplasia were assessed for enrichment in progressing cases versus resolving cases, with an emphasis on genes commonly mutated in EMC. RESULTS: Several mutations were more common in progressing hyperplasia than resolving hyperplasia, although significant overlap was observed between progressing and resolving cases. Mutations included those in PTEN, PIK3CA, and FGFR2, genes commonly mutated in EMC. Mutations in ARID1A and MYC were seen only in progressing hyperplasia, although these were uncommon; this limited diagnostic sensitivity. Progressing hyperplasia demonstrated an accumulation of mutations in oncogenic signaling pathways similarly to endometrial carcinoma. CONCLUSIONS: Because of mutational differences between progressing and nonprogressing hyperplasia, mutational analysis may predict the risk of progression from endometrial hyperplasia to EMC.

Report From the International Society of Urological Pathology (ISUP) Consultation Conference On Molecular Pathology Of Urogenital Cancers. II. Molecular Pathology of Bladder Cancer: Progress and Challenges.

During the 2019 International Society of Urological Pathology Consultation Conference on Molecular Pathology of Urogenital Cancer, the Working Group on Bladder Cancer presented the current status and made recommendations on the diagnostic use of molecular pathology, incorporating a premeeting survey. Bladder cancers are biologically diverse and can be separated into "molecular subtypes," based on expression profiling. These subtypes associate with clinical behavior, histology, and molecular alterations, though their clinical utility has not been demonstrated at present and use in bladder cancer is not recommended. Mutations in the TERT promoter are present in the majority of bladder cancers, including the noninvasive stage of tumor evolution, but not in reactive conditions. Mutational analysis of the TERT promoter thus distinguishes histologically deceptive cancers from their benign mimics in some cases. A minority of pathologists employ this test. FGFR3 mutations are common in bladder cancer, and metastatic urothelial carcinoma (UC) with such mutations frequently responds to erdafitinib, an FGFR inhibitor. Testing for FGFR3 alterations is required before using this drug. Metastatic UC responds to immune-oncology (IO) agents in 20% of cases. These are approved as first and second-line treatments in metastatic UC. Several biological parameters associate with response to IO agents, including tumor mutational burden, molecular subtype, and infiltration by programmed death-ligand 1-positive lymphocytes, detected by immunohistochemistry. Programmed death-ligand 1 immunohistochemistry is mandatory before administering IO agents in the first-line setting. In conclusion, much has been learned about the biology of bladder cancer, and this understanding has improved the care of patients with the disease.

Evaluation of PD-L1 and other immune markers in bladder urothelial carcinoma stratified by histologic variants and molecular subtypes.

Although advanced bladder cancer overall has a poor prognosis, a subset of patients demonstrate durable response to immune checkpoint inhibitors. Evidence shows that the response to checkpoint inhibitors may be associated with type and degree of immune infiltration in the tumor microenvironment. Here, we evaluated immune markers stratified by molecular subtypes and histologic variants. The study utilized a series of urothelial carcinomas (UCs) by tissue microarray, on which histologic variants and molecular subtypes had previously been established. PD1, CD3, CD8 and CD68 expression was evaluated by immunohistochemistry in tumor infiltrating immune cells, while PD-L1 expression in the tumor microenvironment was assessed. Each marker was scored semi-quantitatively (score 0-3). Tumors were clustered by marker scores using agglomerative methods, and associations among markers, histologies, and molecular subtypes were analyzed. PD-L1 expression in the tumor microenvironment significantly correlated with presence of CD3, CD8 and chronic inflammation. Urothelial carcinoma may be classified as either immune high or low based on marker expression. The immune high group is enriched in higher CD3, PD-L1, and genomically-unstable molecular subtype, suggesting it may respond to checkpoint inhibitors. We also identified a degree of intratumoral heterogeneity in immune markers in bladder cancer.

Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer.

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

Repression of transcription factor AP-2 alpha by PPARγ reveals a novel transcriptional circuit in basal-squamous bladder cancer.

The discovery of bladder cancer transcriptional subtypes provides an opportunity to identify high risk patients, and tailor disease management. Recent studies suggest tumor heterogeneity contributes to regional differences in molecular subtype within the tumor, as well as during progression and following treatment. Nonetheless, the transcriptional drivers of the aggressive basal-squamous subtype remain unidentified. As PPARÉ£ has been repeatedly implicated in the luminal subtype of bladder cancer, we hypothesized inactivation of this transcriptional master regulator during progression results in increased expression of basal-squamous specific transcription factors (TFs) which act to drive aggressive behavior. We initiated a pharmacologic and RNA-seq-based screen to identify PPARÉ£-repressed, basal-squamous specific TFs. Hierarchical clustering of RNA-seq data following treatment of three human bladder cancer cells with a PPARÉ£ agonist identified a number of TFs regulated by PPARÉ£ activation, several of which are implicated in urothelial and squamous differentiation. One PPARÉ£-repressed TF implicated in squamous differentiation identified is Transcription Factor Activating Protein 2 alpha (TFAP2A). We show TFAP2A and its paralog TFAP2C are overexpressed in basal-squamous bladder cancer and in squamous areas of cystectomy samples, and that overexpression is associated with increased lymph node metastasis and distant recurrence, respectively. Biochemical analysis confirmed the ability of PPARÉ£ activation to repress TFAP2A, while PPARÉ£ antagonist and PPARÉ£ siRNA knockdown studies indicate the requirement of a functional receptor. In vivo tissue recombination studies show TFAP2A and TFAP2C promote tumor growth in line with the aggressive nature of basal-squamous bladder cancer. Our findings suggest PPARÉ£ inactivation, as well as TFAP2A and TFAP2C overexpression cooperate with other TFs to promote the basal-squamous transition during tumor progression.

Genetic Variants Implicate Dual Oxidase-2 in Familial and Sporadic Nonmedullary Thyroid Cancer.

Highly penetrant hereditary thyroid cancer manifests as familial nonmedullary thyroid cancer (FNMTC), whereas low-penetrance hereditary thyroid cancer manifests as sporadic disease and is associated with common polymorphisms, including rs965513[A]. Whole-exome sequencing of an FNMTC kindred identified a novel Y1203H germline dual oxidase-2 (DUOX2) mutation. DUOX2Y1203H is enzymatically active, with increased production of reactive oxygen species. Furthermore, patients with sporadic thyroid cancer homozygous for rs965513[A] demonstrated higher DUOX2 expression than heterozygous rs965513[A/G] or homozygous rs965513[A]-negative patients. These data suggest that dysregulated hydrogen peroxide metabolism is a common mechanism by which high- and low-penetrance genetic factors increase thyroid cancer risk. SIGNIFICANCE: This study provides novel insights into the genetic and molecular mechanisms underlying familial and sporadic thyroid cancers.

Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.