GPNMB expression identifies TSC1/2/mTOR-associated and MiT family translocation-driven renal neoplasms.

GPNMB (glycoprotein nonmetastatic B) and other TFE3/TFEB transcriptional targets have been proposed as markers for microphthalmia (MiT) translocation renal cell carcinomas (tRCCs). We recently demonstrated that constitutive mTORC1 activation via TSC1/2 loss leads to increased activity of TFE3/TFEB, suggesting that the pathogenesis and molecular markers for tRCCs and TSC1/2-associated tumors may be overlapping. We examined GPNMB expression in human kidney and angiomyolipoma (AML) cell lines with TSC2 and/or TFE3/TFEB loss produced using CRISPR-Cas9 genome editing as well as in a mouse model of Tsc2 inactivation-driven renal tumorigenesis. Using an automated immunohistochemistry (IHC) assay for GPNMB, digital image analysis was employed to quantitatively score expression in clear cell RCC (ccRCC, n = 87), papillary RCC (papRCC, n = 53), chromophobe RCC (chRCC, n = 34), oncocytoma (n = 4), TFE3- or TFEB-driven tRCC (n = 56), eosinophilic solid and cystic RCC (ESC, n = 6), eosinophilic vacuolated tumor (EVT, n = 4), and low-grade oncocytic tumor (LOT, n = 3), as well as AML (n = 29) and perivascular epithelioid cell tumors (PEComas, n = 8). In cell lines, GPNMB was upregulated following TSC2 loss in a MiT/TFE- and mTORC1-dependent fashion. Renal tumors in Tsc2+/- A/J mice showed upregulation of GPNMB compared with normal kidney. Mean GPNMB expression was significantly higher in tRCC than in ccRCC (p < 0.0001), papRCC (p < 0.0001), and chRCC (p < 0.0001). GPNMB expression in TSC1/2/MTOR alteration-associated renal tumors (including ESC, LOT, AML, and PEComa) was comparable to that in tRCC. The immunophenotype of tRCC and TSC1/2/MTOR alteration-associated renal tumors is highly overlapping, likely due to the increased activity of TFE3/TFEB in both, revealing an important caveat regarding the use of TFE3/TFEB-transcriptional targets as diagnostic markers. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Reciprocal YAP1 loss and INSM1 expression in neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is a rare but aggressive histologic variant of prostate cancer that responds poorly to androgen deprivation therapy. Hybrid NEPC-adenocarcinoma (AdCa) tumors are common, often eluding accurate pathologic diagnosis and requiring ancillary markers for classification. We recently performed an outlier-based meta-analysis across a number of independent gene expression microarray datasets to identify novel markers that differentiate NEPC from AdCa, including up-regulation of insulinoma-associated protein 1 (INSM1) and loss of Yes-associated protein 1 (YAP1). Here, using diverse cancer gene expression datasets, we show that Hippo pathway-related genes, including YAP1, are among the top down-regulated gene sets with expression of the neuroendocrine transcription factors, including INSM1. In prostate cancer cell lines, transgenic mouse models, and human prostate tumor cohorts, we confirm that YAP1 RNA and YAP1 protein expression are silenced in NEPC and demonstrate that the inverse correlation of INSM1 and YAP1 expression helps to distinguish AdCa from NEPC. Mechanistically, we find that YAP1 loss in NEPC may help to maintain INSM1 expression in prostate cancer cell lines and we further demonstrate that YAP1 silencing likely occurs epigenetically, via CpG hypermethylation near its transcriptional start site. Taken together, these data nominate two additional markers to distinguish NEPC from AdCa and add to data from other tumor types suggesting that Hippo signaling is tightly reciprocally regulated with neuroendocrine transcription factor expression. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MicroRNA-141-3p regulates cellular proliferation, migration, and invasion in esophageal cancer by targeting tuberous sclerosis complex 1.

MicroRNA (miR)-141-3p, which functions as an oncogene in multiple malignancies, has been shown to be highly overexpressed in esophageal cancer cells in our previous work. miR-141-3p is predicted to bind the messenger RNA (mRNA) of tuberous sclerosis complex 1 (TSC1), a tumor suppressor, with high affinity. In this study, we investigated the expression and functional interaction between miR-141-3p and TSC1 in esophageal cancer cells. Experiments were conducted in four esophageal cancer lines and in tumor cells isolated from human esophageal cancer specimens by laser capture microdissection. miR-141-3p expression was measured by real time and droplet digital PCR. Biotinylated RNA pull-down and luciferase reporter assays were used to assess binding. miR-141-3p function was tested by assessing proliferation, migration, invasion, and induction of autophagy following its silencing. We found that miR-141-3p levels were increased in TE7, OE33, and TE10 esophageal cancer cells compared to FLO-1 cells, with similar heterogeneity observed in human esophageal cancer specimens. Silencing of miR-141-3p led to increased TSC1 protein expression in these cells and was associated with increased TSC1 translation. Binding studies reveal that miR-141-3p binds to each of the predicted binding sites in the 3'-untranslated region of TSC1 mRNA. Following miR-141-3p silencing, TE7, OE33, and TE10 cells exhibited decreased proliferation, migration, and invasion, as well as enhanced autophagy. Importantly, these phenotypic effects were replicated by overexpression of TSC1 alone in these cells. Our results indicate that miR-141-3p functions in an oncogenic capacity in a subset of esophageal cancer cells, in part by suppressing TSC1 expression.

Neuroendocrine differentiation in usual-type prostatic adenocarcinoma: Molecular characterization and clinical significance.

BACKGROUND: Small cell neuroendocrine (NE) carcinomas of the prostate classically lose androgen receptor (AR) expression, may harbor loss of the RB1, TP53, and PTEN tumor suppressor genes, and are associated with a poor prognosis. However usual-type adenocarcinomas may also contain areas of NE differentiation, and in this context the molecular features and biological significance are less certain. METHODS: We examined the molecular phenotype and oncologic outcomes of primary prostate adenocarcinomas with ≥5% NE differentiation (≥5% chromogranin A-positive NE cells in any given tumor spot on tissue microarray) using three independent study sets: a set of tumors with paneth cell-like NE differentiation (n = 26), a retrospective case-cohort of intermediate- and high-risk patients enriched for adverse outcomes (n = 267), and primary tumors from a retrospective series of men with eventual castration-resistant metastatic prostate cancer (CRPC) treated with abiraterone or enzalutamide (n = 55). RESULTS: Benign NE cells expressed significantly lower quantified AR levels compared with paired benign luminal cells (P < .001). Similarly, paneth-like NE carcinoma cells or carcinoma cells expressing chromogranin A expressed significantly lower quantified AR levels than paired non-NE carcinoma cells (P < .001). Quantified ERG protein expression, was also lower in chromogranin A-labeled adenocarcinoma cells compared with unlabeled cells (P < .001) and tumors with NE differentiation showed lower gene expression scores for AR activity compared with those without. Despite evidence of lower AR signaling, adenocarcinomas with NE differentiation did not differ by prevalence of TP53 missense mutations, or PTEN or RB1 loss, compared with those without NE differentiation. Finally, NE differentiation was not associated with time to metastasis in intermediate- and high-risk patients (P = .6 on multivariate analysis), nor with progression-free survival in patients with CRPC treated with abiraterone or enzalutamide (P = .9). CONCLUSION: NE differentiation in usual-type primary prostate adenocarcinoma is a molecularly and clinically distinct form of lineage plasticity from that occurring in small cell NE carcinoma.

Immunolabeling of Cleared Human Pancreata Provides Insights into Three-Dimensional Pancreatic Anatomy and Pathology.

Visualizing pathologies in three dimensions can provide unique insights into the biology of human diseases. A rapid and easy-to-implement dibenzyl ether-based technique was used to clear thick sections of surgically resected human pancreatic parenchyma. Protocols were applicable to both fresh and formalin-fixed, paraffin-embedded tissue. The penetration of antibodies into dense pancreatic parenchyma was optimized using both gradually increasing antibody concentrations and centrifugal flow. Immunolabeling with antibodies against cytokeratin 19 was visualized using both light sheet and confocal laser scanning microscopy. The technique was applied successfully to 26 sections of pancreas, providing three-dimensional (3D) images of normal pancreatic tissue, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, and infiltrating pancreatic ductal adenocarcinomas. 3D visualization highlighted processes that are hard to conceptualize in two dimensions, such as invasive carcinoma growing into what appeared to be pre-existing pancreatic ducts and within venules, and the tracking of long cords of neoplastic cells parallel to blood vessels. Expanding this technique to formalin-fixed, paraffin-embedded tissue opens pathology archives to 3D visualization of unique biosamples and rare diseases. The application of immunolabeling and clearing to human pancreatic parenchyma provides detailed visualization of normal pancreatic anatomy, and can be used to characterize the 3D architecture of diseases including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and pancreatic ductal adenocarcinomas.

Elevated expression of Fn14 in non-small cell lung cancer correlates with activated EGFR and promotes tumor cell migration and invasion.

Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.

An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.

Hyperglycemia regulates RUNX2 activation and cellular wound healing through the aldose reductase polyol pathway.

Diabetes mellitus accelerates cardiovascular microangiopathies and atherosclerosis, which are a consequence of hyperglycemia. The aldose reductase (AR) polyol pathway contributes to these microvascular complications, but how it mediates vascular damage in response to hyperglycemia is less understood. The RUNX2 transcription factor, which is repressed in diabetic animals, promotes vascular endothelial cell (EC) migration, proliferation, and angiogenesis. Here we show that physiological levels of glucose (euglycemia) increase RUNX2 DNA binding and transcriptional activity, whereas hyperglycemia does not. However, inhibition of AR reverses hyperglycemic suppression of RUNX2. IGF-1 secretion and IGF receptor phosphorylation by autocrine IGF-1 occur equally in euglycemic or hyperglycemic conditions, suggesting that reduced RUNX2 activity in response to hyperglycemia is not because of altered IGF-1/IGF receptor activation. AR also negatively regulates RUNX2-dependent vascular remodeling in an EC wounded monolayer assay, which is reversed by specific AR inhibition in hyperglycemia. Thus, euglycemia supports RUNX2 activity and promotes normal microvascular EC migration and wound healing, which are repressed under hyperglycemic conditions through the AR polyol pathway.

mTORC1 feedback to AKT modulates lysosomal biogenesis through MiT/TFE regulation.

The microphthalmia family of transcription factors (MiT/TFEs) controls lysosomal biogenesis and is negatively regulated by the nutrient sensor mTORC1. However, the mechanisms by which cells with constitutive mTORC1 signaling maintain lysosomal catabolism remain to be elucidated. Using the murine epidermis as a model system, we found that epidermal Tsc1 deletion resulted in a phenotype characterized by wavy hair and curly whiskers, and was associated with increased EGFR and HER2 degradation. Unexpectedly, constitutive mTORC1 activation with Tsc1 loss increased lysosomal content via upregulated expression and activity of MiT/TFEs, whereas genetic deletion of Rheb or Rptor or prolonged pharmacologic mTORC1 inactivation had the reverse effect. This paradoxical increase in lysosomal biogenesis by mTORC1 was mediated by feedback inhibition of AKT, and a resulting suppression of AKT-induced MiT/TFE downregulation. Thus, inhibiting hyperactive AKT signaling in the context of mTORC1 loss-of-function fully restored MiT/TFE expression and activity. These data suggest that signaling feedback loops work to restrain or maintain cellular lysosomal content during chronically inhibited or constitutively active mTORC1 signaling, respectively, and reveal a mechanism by which mTORC1 regulates upstream receptor tyrosine kinase signaling.

mTORC1 loss impairs epidermal adhesion via TGF-ß/Rho kinase activation.

Despite its central position in oncogenic intracellular signaling networks, the role of mTORC1 in epithelial development has not been studied extensively in vivo. Here, we have used the epidermis as a model system to elucidate the cellular effects and signaling feedback sequelae of mTORC1 loss of function in epithelial tissue. In mice with conditional epidermal loss of the mTORC1 components Rheb or Rptor, mTORC1 loss of function unexpectedly resulted in a profound skin barrier defect with epidermal abrasions, blistering, and early postnatal lethality, due to a thinned epidermis with decreased desmosomal protein expression and incomplete biochemical differentiation. In mice with mTORC1 loss of function, we found that Rho kinase (ROCK) signaling was constitutively activated, resulting in increased cytoskeletal tension and impaired cell-cell adhesion. Inhibition or silencing of ROCK1 was sufficient to rescue keratinocyte adhesion and biochemical differentiation in these mice. mTORC1 loss of function also resulted in marked feedback upregulation of upstream TGF-ß signaling, triggering ROCK activity and its downstream effects on desmosomal gene expression. These findings elucidate a role for mTORC1 in the regulation of epithelial barrier formation, cytoskeletal tension, and cell adhesion, underscoring the complexity of signaling feedback following mTORC1 inhibition.

The HER2- and heregulin ß1 (HRG)-inducible TNFR superfamily member Fn14 promotes HRG-driven breast cancer cell migration, invasion, and MMP9 expression.

HER2 overexpression occurs in 15% to 20% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Abnormal HER2 activation, either through HER2 overexpression or heregulin (HRG):HER3 binding, elicits the formation of potent HER2-HER3 heterodimers and drives breast cancer cell growth and metastasis. In a previous study, we found that fibroblast growth factor-inducible 14 (Fn14), a member of the TNF receptor superfamily, was frequently overexpressed in human HER2+ breast tumors. We report here that HER2 and Fn14 are also coexpressed in mammary tumors that develop in two different transgenic mouse models of breast cancer. In consideration of these findings, we investigated whether HER2 activation in breast cancer cells could directly induce Fn14 gene expression. We found that transient or stable transfection of MCF7 cells with a HER2 expression plasmid increased Fn14 protein levels. Also, HRG1-ß1 treatment of MCF7 cells transiently induced Fn14 mRNA and protein expression. Both the HER2- and HRG1-ß1-induced increase in Fn14 expression in MCF7 cells as well as basal Fn14 expression in HER2 gene-amplified AU565 cells could be reduced by HER2 kinase inhibition with lapatinib or combined HER2 and HER3 depletion using siRNA. We also report that Fn14-depleted, HER2-overexpressing MCF7 cells have reduced basal cell migration capacity and reduced HRG1-ß1-stimulated cell migration, invasion, and matrix metalloproteinase (MMP)-9 expression. Together, these results indicate that Fn14 may be an important downstream regulator of HER2/HER3-driven breast cancer cell migration and invasion.

The TWEAK receptor Fn14 is a therapeutic target in melanoma: immunotoxins targeting Fn14 receptor for malignant melanoma treatment.

Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in ∼60% of the melanoma cell lines we tested, including both B-Raf WT and B-Raf(V600E) lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested. We generated both a chemical conjugate composed of the recombinant gelonin (rGel) toxin and the anti-Fn14 antibody ITEM-4 (designated ITEM4-rGel) and a humanized, dimeric single-chain antibody of ITEM-4 fused to rGel (designated hSGZ). Both ITEM4-rGel and hSGZ were highly cytotoxic to a panel of different melanoma cell lines. Mechanistic studies showed that both immunotoxins induced melanoma cell necrosis. In addition, these immunotoxins could upregulate the cellular expression of Fn14 and trigger cell-signaling events similar to the Fn14 ligand TWEAK. Finally, treatment of mice bearing human melanoma MDA-MB-435 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared with controls. We conclude that Fn14 is a therapeutic target in melanoma and the hSGZ construct appears to warrant further development as a therapeutic agent against Fn14-positive melanoma.