Genome-wide p63-Target Gene Analyses Reveal TAp63/NRF2-Dependent Oxidative Stress Responses.

The p53 family member TP63 encodes two sets of N-terminal isoforms, TAp63 and ΔNp63 isoforms. They each regulate diverse biological functions in epidermal morphogenesis and in cancer. In the skin, where their activities have been extensively characterized, TAp63 prevents premature aging by regulating the quiescence and genomic stability of stem cells required for wound healing and hair regeneration, while ΔNp63 controls maintenance and terminal differentiation of epidermal basal cells. This functional diversity is surprising given that these isoforms share a high degree of similarity, including an identical sequence for a DNA-binding domain. To understand the mechanisms of the transcriptional programs regulated by each p63 isoform and leading to diverse biological functions, we performed genome-wide analyses using p63 isoform-specific chromatin immunoprecipitation, RNA sequencing, and metabolomics of TAp63-/- and ΔNp63-/- mouse epidermal cells. Our data indicate that TAp63 and ΔNp63 physically and functionally interact with distinct transcription factors for the downstream regulation of their target genes, thus ultimately leading to the regulation of unique transcriptional programs and biological processes. Our findings unveil novel transcriptomes regulated by the p63 isoforms to control diverse biological functions, including the cooperation between TAp63 and NRF2 in the modulation of metabolic pathways and response to oxidative stress providing a mechanistic explanation for the TAp63 knock out phenotypes. SIGNIFICANCE: The p63 isoforms, TAp63 and ΔNp63, control epithelial morphogenesis and tumorigenesis through the interaction with distinct transcription factors and the subsequent regulation of unique transcriptional programs.

Grading of lung adenocarcinomas with simultaneous segmentation by artificial intelligence (GLASS-AI).

Preclinical genetically engineered mouse models (GEMMs) of lung adenocarcinoma are invaluable for investigating molecular drivers of tumor formation, progression, and therapeutic resistance. However, histological analysis of these GEMMs requires significant time and training to ensure accuracy and consistency. To achieve a more objective and standardized analysis, we used machine learning to create GLASS-AI, a histological image analysis tool that the broader cancer research community can utilize to grade, segment, and analyze tumors in preclinical models of lung adenocarcinoma. GLASS-AI demonstrates strong agreement with expert human raters while uncovering a significant degree of unreported intratumor heterogeneity. Integrating immunohistochemical staining with high-resolution grade analysis by GLASS-AI identified dysregulation of Mapk/Erk signaling in high-grade lung adenocarcinomas and locally advanced tumor regions. Our work demonstrates the benefit of employing GLASS-AI in preclinical lung adenocarcinoma models and the power of integrating machine learning and molecular biology techniques for studying the molecular pathways that underlie cancer progression.

Comprehensive Metabolic Tracing Reveals the Origin and Catabolism of Cysteine in Mammalian Tissues and Tumors.

Cysteine plays critical roles in cellular biosynthesis, enzyme catalysis, and redox metabolism. The intracellular cysteine pool can be sustained by cystine uptake or de novo synthesis from serine and homocysteine. Demand for cysteine is increased during tumorigenesis for generating glutathione to deal with oxidative stress. While cultured cells have been shown to be highly dependent on exogenous cystine for proliferation and survival, how diverse tissues obtain and use cysteine in vivo has not been characterized. We comprehensively interrogated cysteine metabolism in normal murine tissues and cancers that arise from them using stable isotope 13C1-serine and 13C6-cystine tracing. De novo cysteine synthesis was highest in normal liver and pancreas and absent in lung tissue, while cysteine synthesis was either inactive or downregulated during tumorigenesis. In contrast, cystine uptake and metabolism to downstream metabolites was a universal feature of normal tissues and tumors. However, differences in glutathione labeling from cysteine were evident across tumor types. Thus, cystine is a major contributor to the cysteine pool in tumors, and glutathione metabolism is differentially active across tumor types. SIGNIFICANCE: Stable isotope 13C1-serine and 13C6-cystine tracing characterizes cysteine metabolism in normal murine tissues and its rewiring in tumors using genetically engineered mouse models of liver, pancreas, and lung cancers.

Pramlintide: A Novel Therapeutic Approach for Osteosarcoma through Metabolic Reprogramming.

Despite aggressive combination chemotherapy and surgery, outcomes for patients with osteosarcoma have remained stagnant for more than 25 years, and numerous clinical trials have identified no new therapies. p53 deletion or mutation is found in more than 80% of osteosarcoma tumors. In p53-deficient cancers with structurally altered p63 and p73, interfering with tumor cell metabolism using Pramlintide (an FDA-approved drug for type 2 diabetes) results in tumor regression. Pramlintide response is mediated through upregulation of islet amyloid polypeptide (IAPP). Here, we showed that osteosarcoma cells have altered p63, p73, and p53, and decreased IAPP expression but have the two main IAPP receptors, CalcR and RAMP3, which inhibit glycolysis and induce apoptosis. We showed that in osteosarcoma cells with high- or mid-range glycolytic activity, Pramlintide decreased cell glycolysis, resulting in decreased proliferation and increased apoptosis in vitro. In contrast, Pramlintide had no effect in osteosarcoma cells with low glycolytic activity. Using a subcutaneous osteosarcoma mouse model, we showed that intratumoral injection of Pramlintide-induced tumor regression. Tumor sections showed increased apoptosis and a decrease in Ki-67 and HIF-1α. These data suggest that in osteosarcoma cells with altered p53, p63, and p73 and a high glycolytic function, Pramlintide therapy can modulate metabolic programming and inhibit tumor growth.

Clinical Trial Development in TP53-Mutated Locally Advanced and Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma.

TP53 mutation is the most frequent genetic event in head and neck squamous cell carcinoma (HNSCC), found in more than 80% of patients with human papillomavirus-negative disease. As mutations in the TP53 gene are associated with worse outcomes in HNSCC, novel therapeutic approaches are needed for patients with TP53-mutated tumors. The National Cancer Institute sponsored a Clinical Trials Planning Meeting to address the issues of identifying and developing clinical trials for patients with TP53 mutations. Subcommittees, or breakout groups, were tasked with developing clinical studies in both the locally advanced and recurrent and/or metastatic (R/M) disease settings as well as considering signal-seeking trial designs. A fourth breakout group was focused on identifying and standardizing biomarker integration into trial design; this information was provided to the other breakout groups prior to the meeting to aid in study development. A total of 4 concepts were prioritized to move forward for further development and implementation. This article summarizes the proceedings of the Clinical Trials Planning Meeting with the goal of developing clinical trials for patients with TP53-mutant HNSCC that can be conducted within the National Clinical Trials Network.

p63, a key regulator of Ago2, links to the microRNA-144 cluster.

As a key component of the RNA-induced silencing complex (RISC), Argonaute2 (Ago2) exhibits a dual function regulatory role in tumor progression. However, the mechanistic basis of differential regulation remains elusive. p63 is a homolog of the tumor suppressor p53. p63 isoforms play a critical role in tumorigenesis and metastasis. Herein, we show that p63 isoforms physically interact with and stabilize Ago2. Expression of p63 isoforms increases the levels of Ago2 protein, while depletion of p63 isoforms by shRNA decreases Ago2 protein levels. p63 strongly guides Ago2 dual functions in vitro and in vivo. Ectopic expression of the miR-144/451 cluster increases p63 protein levels; TAp63 transactivates the miR-144/451 cluster, forming a positive feedback loop. Notably, miR-144 activates p63 by directly targeting Itch, an E3 ligase of p63. Ectopic expression of miR-144 induces apoptosis in H1299 cells. miR-144 enhances TAp63 tumor suppressor function and inhibits cell invasion. Our findings uncover a novel function of p63 linking the miRNA-144 cluster and the Ago2 pathway. FACTS AND QUESTIONS: Identification of Ago2 as a p63 target. Ago2 exhibits a dual function regulatory role in tumor progression; however, the molecular mechanism of Ago2 regulation remains unknown. p63 strongly guides Ago2 dual functions in vitro and in vivo. Unraveling a novel function of p63 links the miRNA-144 cluster and the Ago2 pathway.

ΔNp63 regulates a common landscape of enhancer associated genes in non-small cell lung cancer.

Distinct lung stem cells give rise to lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). ΔNp63, the p53 family member and p63 isoform, guides the maturation of these stem cells through the regulation of their self-renewal and terminal differentiation; however, the underlying mechanistic role regulated by ∆Np63 in lung cancer development has remained elusive. By utilizing a ΔNp63-specific conditional knockout mouse model and xenograft models of LUAD and LUSC, we found that ∆Np63 promotes non-small cell lung cancer by maintaining the lung stem cells necessary for lung cancer cell initiation and progression in quiescence. ChIP-seq analysis of lung basal cells, alveolar type 2 (AT2) cells, and LUAD reveals robust ∆Np63 regulation of a common landscape of enhancers of cell identity genes. Importantly, one of these genes, BCL9L, is among the enhancer associated genes regulated by ∆Np63 in Kras-driven LUAD and mediates the oncogenic effects of ∆Np63 in both LUAD and LUSC. Accordingly, high BCL9L levels correlate with poor prognosis in LUAD patients. Taken together, our findings provide a unifying oncogenic role for ∆Np63 in both LUAD and LUSC through the regulation of a common landscape of enhancer associated genes.

miR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFßR3.

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFß signaling, TGFßR3. miR-181a and TGFßR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFßR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFßR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFßR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFßR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFß2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm in vivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFßR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

The Landmark Discovery That Paved the Way to a Mechanistic Understanding of P53 Gain of Function and Personalized Medicine.

In 1990, Baker and colleagues reported their seminal findings in Cancer Research focusing on the transition from adenoma to carcinoma of the colon. By sequencing the TP53 locus in 58 colorectal tumors (25 adenomas and 33 carcinomas) and measuring its allelic deletions, they discovered that this transition requires the loss of one TP53 allele and the mutation of the other one. Here, we discuss how this landmark discovery shed a new light on p53 mutations, prompting the generation of novel mouse models that definitively proved the mutant p53 gain-of-function hypothesis suggested by these results. Finally, we evaluate the implications that the Vogelstein model of cancer progression had on numerous aspects of cancer biology and cancer care, including the characterization of tumor evolution and the response to therapy, and how it ultimately contributed to the wider adoption of early detection screenings and personalized medicine.See related article by Baker and colleagues, Cancer Res 1990;50:7717-22.

Deep Learning of Histopathology Images at the Single Cell Level.

The tumor immune microenvironment (TIME) encompasses many heterogeneous cell types that engage in extensive crosstalk among the cancer, immune, and stromal components. The spatial organization of these different cell types in TIME could be used as biomarkers for predicting drug responses, prognosis and metastasis. Recently, deep learning approaches have been widely used for digital histopathology images for cancer diagnoses and prognoses. Furthermore, some recent approaches have attempted to integrate spatial and molecular omics data to better characterize the TIME. In this review we focus on machine learning-based digital histopathology image analysis methods for characterizing tumor ecosystem. In this review, we will consider three different scales of histopathological analyses that machine learning can operate within: whole slide image (WSI)-level, region of interest (ROI)-level, and cell-level. We will systematically review the various machine learning methods in these three scales with a focus on cell-level analysis. We will provide a perspective of workflow on generating cell-level training data sets using immunohistochemistry markers to "weakly-label" the cell types. We will describe some common steps in the workflow of preparing the data, as well as some limitations of this approach. Finally, we will discuss future opportunities of integrating molecular omics data with digital histopathology images for characterizing tumor ecosystem.

Elective neck dissection versus observation in patients with head and neck cutaneous squamous cell carcinoma.

BACKGROUND: The survival benefit of elective neck dissection (END) for patients with cutaneous squamous cell carcinoma (cSCC) of the head and neck and no evidence of regional metastasis (cN0) has never been reported. The aim of this study was to determine the effect of END on patient survival. METHODS: The authors included patients with head and neck cSCC who had undergone primary surgery from 1995 to 2017. The primary end point was survival, and the secondary end points were the incidence of occult regional disease and regional disease control. To assess the impact of END on survival, the authors used multivariable Cox proportional hazards models with propensity score and matching techniques for internal validation. RESULTS: A total of 1111 patients presented with no evidence of nodal disease; 173 had END, and 938 were observed. Adjuvant radiotherapy to the neck was administered to 101 patients (9%). END resulted in a 5-year overall survival rate of 52%, whereas the rate was 63% in the observation group (P = .003 [log-rank]). The 5-year disease-free survival rate for patients undergoing END was similar to that for the observation group (73% vs 75%; P = .429). A multivariate regression model showed that the performance of END was not associated with improved rates of overall, disease-specific, or disease-free survival; similarly, among patients with advanced disease (T3-4), those who underwent END did not have improved survival rates. CONCLUSIONS: Among patients with cSCC of the head and neck, observation of the neck nodes resulted in noninferior survival rates in comparison with END at the time of primary surgery. Further studies are required to elucidate the role of END in patients with advanced disease.

Mutant p53s and chromosome 19 microRNA cluster overexpression regulate cancer testis antigen expression and cellular transformation in hepatocellular carcinoma.

A subset of hepatocellular carcinoma (HCC) overexpresses the chromosome 19 miRNA cluster (C19MC) and is associated with an undifferentiated phenotype marked by overexpression of cancer testis antigens (CTAs) including anti-apoptotic melanoma-A antigens (MAGEAs). However, the regulation of C19MC miRNA and MAGEA expression in HCCs are not understood. Here we show that, C19MC overexpression is tightly linked to a sub-set of HCCs with transcription-incompetent p53. Using next-generation and Sanger sequencing we found that, p53 in Hep3B cells is impaired by TP53-FXR2 fusion, and that overexpression of the C19MC miRNA-520G in Hep3B cells promotes the expression of MAGEA-3, 6 and 12 mRNAs. Furthermore, overexpression of p53-R175H and p53-R273H mutants promote miR-520G and MAGEA RNA expression and cellular transformation. Moreover, IFN-γ co-operates with miR-520G to promote MAGEA expression. On the other hand, metals such as nickel and zinc promote miR-526B but not miR-520G, to result in the suppression of MAGEA mRNA expression, and evoke cell death through mitochondrial membrane depolarization. Therefore our study demonstrates that a MAGEA-promoting network involving miR-520G, p53-defects and IFN-γ that govern cellular transformation and cell survival pathways, but MAGEA expression and survival are counteracted by nickel and zinc combination.

TIMEx: tumor-immune microenvironment deconvolution web-portal for bulk transcriptomics using pan-cancer scRNA-seq signatures.

SUMMARY: The heterogeneous cell types of the tumor-immune microenvironment (TIME) play key roles in determining cancer progression, metastasis and response to treatment. We report the development of TIMEx, a novel TIME deconvolution method emphasizing on estimating infiltrating immune cells for bulk transcriptomics using pan-cancer single-cell RNA-seq signatures. We also implemented a comprehensive, user-friendly web-portal for users to evaluate TIMEx and other deconvolution methods with bulk transcriptomic profiles. AVAILABILITY AND IMPLEMENTATION: TIMEx web-portal is freely accessible at http://timex.moffitt.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Hsp70 acts as a fine-switch that controls E3 ligase CHIP-mediated TAp63 and ΔNp63 ubiquitination and degradation.

The major clinical problem in human cancer is metastasis. Metastases are the cause of 90% of human cancer deaths. TAp63 is a critical suppressor of tumorigenesis and metastasis. ΔNp63 acts as a dominant-negative inhibitor to block the function of p53 and TAp63. Although several ubiquitin E3 ligases have been reported to regulate p63 stability, the mechanism of p63 regulation remains partially understood. Herein, we show that CHIP, an E3 ligase with a U-box domain, physically interacts with p63 and promotes p63 degradation. Notably, Hsp70 depletion by siRNA stabilizes TAp63 in H1299 cells and destabilizes ΔNp63 in SCC9 cells. Loss of Hsp70 results in a reduction in the TAp63-CHIP interaction in H1299 cells and an increase in the interaction between ΔNp63 and CHIP in SCC9 cells. Our results reveal that Hsp70 acts as a molecular switch to control CHIP-mediated ubiquitination and degradation of p63 isoforms. Furthermore, regulation of p63 by the Hsp70-CHIP axis contributes to the migration and invasion of tumor cells. Hence, our findings demonstrate that Hsp70 is a crucial regulator of CHIP-mediated ubiquitination and degradation of p63 isoforms and identify a new pathway for maintaining TAp63 or ΔNp63 stability in cancers.

Thermodynamic Model for the Design of a Process of Production of Copper Sulfate Pentahydrate from Copper Ores.

In Chile, one of the ways in which small-scale mining industries sustain themselves is through the sale of copper ores to the state company ENAMI, which monetizes this product depending on the copper's mineral grade. To sell this mineral, small mining companies must transport the product to ENAMI, which means a high monetary cost, added to the fact that there are large amounts of waste minerals that cannot be sold because of their low grade. The present work aims that small miners can process these copper ores in situ to commercialize a more valuable product, such as copper salts. Considering the high solar radiation and the scarce superficial water resources found in the north side of the country, a possible process alternative is the leaching of the ores using acid seawater solutions followed by crystallization by solar evaporation. As a necessary tool for this process design, the present work has developed a model able to predict the copper sulfate pentahydrate crystallization from multicomponent solutions, preventing the co-precipitation of undesired compounds (such as iron salts, sodium chloride, and sodium sulphate among others) that contaminate the final product. The Pitzer thermodynamic model was successfully applied to predict the crystallization process of copper sulfate pentahydrate from synthetic leaching solutions. These results were validated through experimental tests.

Pan-cancer analysis reveals TAp63-regulated oncogenic lncRNAs that promote cancer progression through AKT activation.

The most frequent genetic alterations across multiple human cancers are mutations in TP53 and the activation of the PI3K/AKT pathway, two events crucial for cancer progression. Mutations in TP53 lead to the inhibition of the tumour and metastasis suppressor TAp63, a p53 family member. By performing a mouse-human cross species analysis between the TAp63 metastatic mammary adenocarcinoma mouse model and models of human breast cancer progression, we identified two TAp63-regulated oncogenic lncRNAs, TROLL-2 and TROLL-3. Further, using a pan-cancer analysis of human cancers and multiple mouse models of tumour progression, we revealed that these two lncRNAs induce the activation of AKT to promote cancer progression by regulating the nuclear to cytoplasmic translocation of their effector, WDR26, via the shuttling protein NOLC1. Our data provide preclinical rationale for the implementation of these lncRNAs and WDR26 as therapeutic targets for the treatment of human tumours dependent upon mutant TP53 and/or the PI3K/AKT pathway.

Integrative transcriptomic analysis for linking acute stress responses to squamous cell carcinoma development.

Cutaneous squamous cell carcinoma (cuSCC) is the second most common skin cancer and commonly arises in chronically UV-exposed skin or chronic wounds. Since UV exposure and chronic wounds are the two most prominent environmental factors that lead to cuSCC initiation, we undertook this study to test whether more acute molecular responses to UV and wounding overlapped with molecular signatures of cuSCC. We reasoned that transcriptional signatures in common between acutely UV-exposed skin, wounded skin, and cuSCC tumors, might enable us to identify important pathways contributing to cuSCC. We performed transcriptomic analysis on acutely UV-exposed human skin and integrated those findings with datasets from wounded skin and our transcriptomic data on cuSCC using functional pair analysis, GSEA, and pathway analysis. Integrated analyses revealed significant overlap between these three datasets, thus highlighting deep molecular similarities these biological processes, and we identified Oncostatin M (OSM) as a potential common upstream driver. Expression of OSM and its downstream targets correlated with poorer overall survival in head and neck SCC patients. In vitro, OSM promoted invasiveness of keratinocytes and cuSCC cells and suppressed apoptosis of irradiated keratinocytes. Together, these results support the concept of using an integrated, biologically-informed approach to identify potential promoters of tumorigenesis.