ß-catenin, Pax2, and Pten Panel Identifies Precancers Among Histologically Subdiagnostic Endometrial Lesions.

Despite refinements in histologic criteria for the diagnosis of endometrioid precancers, many challenging cases are encountered in daily practice, creating diagnostic uncertainty and suboptimal patient management. Recently, an immunohistochemical 3-marker panel consisting of ß-catenin, Pax2, and Pten was identified as a useful diagnostic adjunct. However, previous studies focused either on cancers or diagnostically unambiguous precancers, leaving questions about the applicability and utility of the panel in endometria with architectural features near or below the threshold of accepted histologic criteria for endometrioid precancers. Here, in a retrospective study of 90 patients, we evaluated the performance of the 3-marker panel. Notably, the panel detected a subset of disordered proliferative endometria (8/44, 18%), nonatypical hyperplasias (19/40, 48%), and cases with ambiguous features (3/6, 50%) with aberrancy for ≥1 markers. Marker-aberrant cases were more likely to progress to endometrioid precancer or cancer ( P =0.0002). Patterns of marker aberrancy in the index and progressor cases from individual patients provided evidence for origin in a common precursor, and next-generation sequencing of the progressor cases rationalized marker aberrancy for ß-catenin and Pten. The results unequivocally demonstrate that some lesions that do not approach current histologic thresholds are bona fide neoplastic precursors with clinically-relevant driver events that can be detected by the 3-marker panel. The findings provide further validation for the diagnostic utility of the panel in clinical practice and its application in difficult or ambiguous cases.

Loss of liver kinase B1 in human seminoma.

Testicular cancer is a common malignancy of young males and is believed to be originated from defective embryonic or adult germ cells. Liver kinase B1 (LKB1) is a serine/threonine kinase and a tumor suppressor gene. LKB1 is a negative regulator of the mammalian target of rapamycin (mTOR) pathway, often inactivated in many human cancer types. In this study, we investigated the involvement of LKB1 in the pathogenesis of testicular germ cell cancer. We performed immunodetection of LKB1 protein in human seminoma samples. A 3D culture model of human seminoma was developed from TCam-2 cells, and two mTOR inhibitors were tested for their efficacy against these cancer cells. Western blot and mTOR protein arrays were used to show that these inhibitors specifically target the mTOR pathway. Examination of LKB1 showed reduced expression in germ cell neoplasia in situ lesions and seminoma compared to adjacent normal-appearing seminiferous tubules where the expression of this protein was present in the majority of germ cell types. We developed a 3D culture model of seminoma using TCam-2 cells, which also showed reduced levels of LKB1 protein. Treatment of TCam-2 cells in 3D with two well-known mTOR inhibitors resulted in reduced proliferation and survival of TCam-2 cells. Overall, our results support that downregulation or loss of LKB1 marks the early stages of the pathogenesis of seminoma, and the suppression of downstream signaling to LKB1 might be an effective therapeutic strategy against this cancer type.

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super-enhancers in maintaining EBV latency.

We probed the lifecycle of Epstein-Barr virus (EBV) on a cell-by-cell basis using single cell RNA sequencing (scRNA-seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350- LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1-α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1-α signaling, markedly induced this cluster. The second cluster, containing GP350+ LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super-enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+ LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE-regulated genes. Furthermore, CRISPR-mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV-associated heterogeneity among LCLs that may have functional consequence on host and viral biology.

Endometrial polyps are non-neoplastic but harbor epithelial mutations in endometrial cancer drivers at low allelic frequencies.

Endometrial polyps (EMPs) are common exophytic masses associated with abnormal uterine bleeding and infertility. Unlike normal endometrium, which is cyclically shed, EMPs persist over ovulatory cycles and after the menopause. Despite their usual classification as benign entities, EMPs are paradoxically associated with endometrial carcinomas of diverse histologic subtypes, which frequently arise within EMPs. The etiology and potential origins of EMPs as clonally-derived neoplasms are uncertain, but previous investigations suggested that EMPs are neoplasms of stromal origin driven by recurring chromosomal rearrangements. To better define benign EMPs at the molecular genetic level, we analyzed individual EMPs from 31 women who underwent hysterectomy for benign indications. The 31 EMPs were subjected to comprehensive genomic profiling by exome sequencing of a large panel of tumor-related genes including oncogenes, tumor suppressors, and chromosomal translocation partners. There were no recurring chromosomal rearrangements, and copy-number analyses did not reveal evidence of significant chromosome-level events. Surprisingly, there was a high incidence of single nucleotide variants corresponding to classic oncogenic drivers (i.e., definitive cancer drivers). The spectrum of known oncogenic driver events matched that of endometrial cancers more closely than any other common cancer. Further analyses including laser-capture microdissection showed that these mutations were present in the epithelial compartment at low allelic frequencies. These results establish a link between EMPs and the acquisition of endometrial cancer driver mutations. Based on these findings, we propose a model where the association between EMPs and endometrial cancer is explained by the age-related accumulation of endometrial cancer drivers in a protected environment that-unlike normal endometrium-is not subject to cyclical shedding.

FOXA2 suppresses endometrial carcinogenesis and epithelial-mesenchymal transition by regulating enhancer activity.

FOXA2 encodes a transcription factor mutated in 10% of endometrial cancers (ECs), with a higher mutation rate in aggressive variants. FOXA2 has essential roles in embryonic and uterine development. However, FOXA2's role in EC is incompletely understood. Functional investigations using human and mouse EC cell lines revealed that FOXA2 controls endometrial epithelial gene expression programs regulating cell proliferation, adhesion, and endometrial-epithelial transition. In live animals, conditional inactivation of Foxa2 or Pten alone in endometrial epithelium did not result in ECs, but simultaneous inactivation of both genes resulted in lethal ECs with complete penetrance, establishing potent synergism between Foxa2 and PI3K signaling. Studies in tumor-derived cell lines and organoids highlighted additional invasion and cell growth phenotypes associated with malignant transformation and identified key mediators, including Myc and Cdh1. Transcriptome and cistrome analyses revealed that FOXA2 broadly controls gene expression programs through modification of enhancer activity in addition to regulating specific target genes, rationalizing its tumor suppressor functions. By integrating results from our cell lines, organoids, animal models, and patient data, our findings demonstrated that FOXA2 is an endometrial tumor suppressor associated with aggressive disease and with shared commonalities among its roles in endometrial function and carcinogenesis.

Reliable Identification of Endometrial Precancers Through Combined Pax2, ß-Catenin, and Pten Immunohistochemistry.

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably ß-catenin and Arid1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n=111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, ß-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n=79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), ß-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or ß-catenin. A focused panel of only 3 markers (Pax2, Pten, and ß-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.

Serial genomic analysis of endometrium supports the existence of histologically indistinct endometrial cancer precursors.

The endometrium is unique as an accessible anatomic location that can be repeatedly biopsied and where diagnostic biopsies do not extirpate neoplastic lesions. We exploited these features to retrospectively characterize serial genomic alterations along the precancer/cancer continuum in individual women. Cases were selected based on (1) endometrial cancer diagnosis/hysterectomy and (2) preceding serial endometrial biopsies including for some patients an early biopsy before a precancer histologic diagnosis. A comprehensive panel was designed for endometrial cancer genes. Formalin-fixed, paraffin-embedded specimens for each cancer, preceding biopsies, and matched germline samples were subjected to barcoded high-throughput sequencing to identify mutations and track their origin and allelic frequency progression. In total, 92 samples from 21 patients were analyzed, providing an opportunity for new insights into early endometrial cancer progression. Definitive invasive endometrial cancers exhibited expected mutational spectra, and canonical driver mutations were detectable in preceding biopsies. Notably, ≥1 cancer mutations were detected prior to the histopathologic diagnosis of an endometrial precancer in the majority of patients. In 18/21 cases, ≥1 mutations were confirmed by abnormal protein levels or subcellular localization by immunohistochemistry, confirming genomic data and providing unique views of histologic correlates. In 19 control endometria, mutation counts were lower, with a lack of canonical endometrial cancer hotspot mutations. Our study documents the existence of endometrial lesions that are histologically indistinct but are bona fide endometrial cancer precursors. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca2+-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca2+ imaging that TRPV1 regulates Ca2+ influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.

A PoleP286R mouse model of endometrial cancer recapitulates high mutational burden and immunotherapy response.

Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.

Fbxw7 is a driver of uterine carcinosarcoma by promoting epithelial-mesenchymal transition.

Uterine carcinosarcoma is an aggressive variant of endometrial carcinoma characterized by unusual histologic features including discrete malignant epithelial and mesenchymal components (carcinoma and sarcoma). Recent studies have confirmed a monoclonal origin, and comprehensive genomic characterizations have identified mutations such as Tp53 and Pten However, the biological origins and specific combination of driver events underpinning uterine carcinosarcoma have remained mysterious. Here, we explored the role of the tumor suppressor Fbxw7 in endometrial cancer through defined genetic model systems. Inactivation of Fbxw7 and Pten resulted in the formation of precancerous lesions (endometrioid intraepithelial neoplasia) and well-differentiated endometrioid adenocarcinomas. Surprisingly, all adenocarcinomas eventually developed into definitive uterine carcinosarcomas with carcinomatous and sarcomatous elements including heterologous differentiation, yielding a faithful genetically engineered model of this cancer type. Genomic analysis showed that most tumors spontaneously acquired Trp53 mutations, pointing to a triad of pathways (p53, PI3K, and Fbxw7) as the critical combination underpinning uterine carcinosarcoma, and to Fbxw7 as a key driver of this enigmatic endometrial cancer type. Lineage tracing provided formal genetic proof that the uterine carcinosarcoma cell of origin is an endometrial epithelial cell that subsequently undergoes a prominent epithelial-mesenchymal transition underlying the attainment of a highly invasive phenotype specifically driven by Fbxw7.

The Emerging Role of the Microenvironment in Endometrial Cancer.

Endometrial cancer (EC) is one of the most frequently diagnosed cancers in women, and despite recent therapeutic advances, in many cases, treatment failure results in cancer recurrence, metastasis, and death. Current research demonstrates that the interactive crosstalk between two discrete cell types (tumor and stroma) promotes tumor growth and investigations have uncovered the dual role of the stromal cells in the normal and cancerous state. In contrast to tumor cells, stromal cells within the tumor microenvironment (TME) are genetically stable. However, tumor cells modify adjacent stromal cells in the TME. The alteration in signaling cascades of TME from anti-tumorigenic to pro-tumorigenic enhances metastatic potential and/or confers therapeutic resistance. Therefore, the TME is a fertile ground for the development of novel therapies. Furthermore, disrupting cancer-promoting signals from the TME or re-educating stromal cells may be an effective strategy to impair metastatic progression. Here, we review the paradoxical role of different non-neoplastic stromal cells during specific stages of EC progression. We also suggest that the inhibition of microenvironment-derived signals may suppress metastatic EC progression and offer novel potential therapeutic interventions.

Adipose-Derived VEGF-mTOR Signaling Promotes Endometrial Hyperplasia and Cancer: Implications for Obese Women.

Obesity is responsible for increased morbidity and mortality in endometrial cancer. Despite the positive correlation of body mass index (BMI) or obesity in endometrial carcinogenesis, the contribution of adipose tissue to the pathogenesis of endometrial hyperplasia and cancer is unclear. This study clarifies the role of adipocytes in the pathogenesis of endometrial cancer by demonstrating that adipocyte-conditioned medium (ACM) increases proliferation, migration, and survival of endometrial cancer cells compared with preadipocyte-conditioned medium (PACM). Comparative cytokine array analysis of ACM and PACM reveal upregulation of a group of cytokines belonging to the VEGF signaling pathway in ACM. VEGF protein expression is upregulated in visceral adipose tissue (VAT) in obese patients, which is correlated with increased tumor growth in an in vivo xenograft model. The increased tumor size is mechanistically associated with the activation of the PI3K/AKT/mTOR pathway, a downstream target of VEGF signaling, and its suppression decreased the growth-promoting effects of VAT on endometrial cancer cells. Similar to the human model systems, pathologic changes in endometrial cells in a hyperphagic obese mouse model are associated with increased body weight and hyperactive mTOR signaling. Analysis of human tissue specimens depicts increased in tumor vasculature and VEGF-mTOR activity in obese endometrial cancer patients compared with nonobese patients. Collectively, these results provide evidence that VEGF-mTOR signaling drives endometrial cell growth leading to hyperplasia and cancer.Implications: Adipocyte-derived VEGF-mTOR signaling may be an attractive therapeutic target against endometrial cancer in obese women. Mol Cancer Res; 16(2); 309-21. ©2017 AACR.

Inhibition of extracellular matrix mediated TGF-ß signalling suppresses endometrial cancer metastasis.

Although aggressive invasion and distant metastases are an important cause of morbidity and mortality in patients with endometrial cancer (EC), the requisite events determining this propensity are currently unknown. Using organotypic three-dimensional culture of endometrial cancer cell lines, we demonstrated anti-correlated TGF-ß signalling gene expression patterns that arise among extracellular matrix (ECM)-attached cells. TGF-ß pathway seemed to be active in EC cells forming non-glandular colonies in 3D-matrix but weaker in glandular colonies. Functionally we found that out of several ECM proteins, fibronectin relatively promotes Smad phosphorylation suggesting a potential role in regulating TGF-ß signalling in non-glandular colonies. Importantly, alteration of TGF-ß pathway induced EMT and MET in both type of colonies through slug protein. The results exemplify a crucial role of TGF-ß pathway during EC metastasis in human patients and inhibition of the pathway in a murine model impaired tumour cell invasion and metastasis depicting an attractive target for therapeutic intervention of malignant tumour progression. These findings provide key insights into the role of ECM-derived TGF-ß signalling to promote endometrial cancer metastasis and offer an avenue for therapeutic targeting of microenvironment derived signals along with tumour cells.

Regulation of Viral Replication, Apoptosis and Pro-Inflammatory Responses by 17-AAG during Chikungunya Virus Infection in Macrophages.

Chikungunya virus (CHIKV) infection has re-emerged as a major public health concern due to its recent worldwide epidemics and lack of control measures. Although CHIKV is known to infect macrophages, regulation of CHIKV replication, apoptosis and immune responses towards macrophages are not well understood. Accordingly, the Raw264.7 cells, a mouse macrophage cell line, were infected with CHIKV and viral replication as well as new viral progeny release was assessed by flow cytometry and plaque assay, respectively. Moreover, host immune modulation and apoptosis were studied through flow cytometry, Western blot and ELISA. Our current findings suggest that expression of CHIKV proteins were maximum at 8 hpi and the release of new viral progenies were remarkably increased around 12 hpi. The induction of Annexin V binding, cleaved caspase-3, cleaved caspase-9 and cleaved caspase-8 in CHIKV infected macrophages suggests activation of apoptosis through both intrinsic and extrinsic pathways. The pro-inflammatory mediators (TNF and IL-6) MHC-I/II and B7.2 (CD86) were also up-regulated during infection over time. Further, 17-AAG, a potential HSP90 inhibitor, was found to regulate CHIKV infection, apoptosis and pro-inflammatory cytokine/chemokine productions of host macrophages significantly. Hence, the present findings might bring new insight into the therapeutic implication in CHIKV disease biology.

Age related increase in mTOR activity contributes to the pathological changes in ovarian surface epithelium.

Ovarian cancer is a disease of older women. However, the molecular mechanisms of ovarian aging and their contribution to the pathogenesis of ovarian cancer are currently unclear. mTOR signalling is a major regulator of aging as suppression of this pathway extends lifespan in model organisms. Overactive mTOR signalling is present in up to 80% of ovarian cancer samples and is associated with poor prognosis. This study examined the role of mTOR signalling in age-associated changes in ovarian surface epithelium (OSE). Histological examination of ovaries from both aged mice and women revealed OSE cell hyperplasia, papillary growth and inclusion cysts. These pathological lesions expressed bonafide markers of ovarian cancer precursor lesions, Pax8 and Stathmin 1, and were presented with elevated mTOR signalling. To understand whether overactive mTOR signalling is responsible for the development of these pathological changes, we analysed ovaries of the Pten trangenic mice and found significant reduction in OSE lesions compared to controls. Furthermore, pharmacological suppression of mTOR signalling significantly decreased OSE hyperplasia in aged mice. Treatment with mTOR inhibitors reduced human ovarian cancer cell viability, proliferation and colony forming ability. Collectively, we have established the role of mTOR signalling in age-related OSE pathologies and initiation of ovarian cancer.

Functional expression of TRPV channels in T cells and their implications in immune regulation.

The importance of Ca(2+) signalling and temperature in the context of T cell activation is well known. However, the molecular identities of key players involved in such critical regulations are still unknown. In this work we explored the endogenous expression of transient receptor potential vanilloid (TRPV) channels, a group of thermosensitive and non-selective cation channels, in T cells. Using flow cytometry and confocal microscopy, we demonstrate that members belonging to the TRPV subfamily are expressed endogenously in the human T cell line Jurkat, in primary human T cells and in primary murine splenic T cells. We also demonstrate that TRPV1- and TRPV4-specific agonists, namely resiniferatoxin and 4α-phorbol-12,13-didecanoate, can cause Ca(2+) influx in T cells. Moreover, our results show that expression of these channels can be upregulated in T cells during concanavalin A-driven mitogenic and anti-CD3/CD28 stimulated TCR activation of T cells. By specific blocking of TRPV1 and TRPV4 channels, we found that these TRPV inhibitors may regulate mitogenic and T cell receptor mediated T cell activation and effector cytokine(s) production by suppressing tumour necrosis factor, interleukin-2 and interferon-γ release. These results may have broad implications in the context of cell-mediated immunity, especially T cell responses and their regulations, neuro-immune interactions and molecular understanding of channelopathies.

Mammalian non-classical major histocompatibility complex I and its receptors: Important contexts of gene, evolution, and immunity.

The evolutionary conserved, less-polymorphic, nonclassical major histocompatibility complex (MHC) class I molecules: Qa-1 and its human homologue human leukocyte antigen-E (HLA-E) along with HLA-F, G and H cross-talk with the T-cell receptors and also interact with natural killer T-cells and other lymphocytes. Moreover, these nonclassical MHC molecules are known to interact with CD94/NKG2 heterodimeric receptors to induce immune responses and immune regulations. This dual role of Qa-1/HLA-E in terms of innate and adaptive immunity makes them more interesting. This review highlights the new updates of the mammalian nonclassical MHC-I molecules in terms of their gene organization, evolutionary perspective and their role in immunity.