Cancer-Associated Fibroblasts in Esophageal Cancer.

Cancer-associated fibroblasts (CAFs), a heterogenous population, can promote cancer cell proliferation, migration, invasion, immunosuppression, and therapeutic resistance in solid tumors. These effects are mediated through secretion of cytokines and growth factors, remodeling of the extracellular matrix, and providing metabolic support for cancer cells. The presence of CAFs in esophageal carcinoma are associated with reduced overall survival and increased resistance to chemotherapy and radiotherapy; thus, identifying therapeutic vulnerabilities of CAFs is a necessity. In esophageal cancer, the mechanisms for CAF recruitment, CAF-mediated promotion of tumorigenesis, metastatic dissemination, and therapeutic resistance have yet to be fully evaluated. Here, we provide an overview of the current understanding of CAFs in esophageal cancer, namely in esophageal squamous cell carcinoma and esophageal adenocarcinoma, as well as in the preneoplastic conditions that predispose to these cancers. Interestingly, there is a discrepancy in our knowledge of CAF biology between esophageal cancer subtypes, with very few studies in esophageal adenocarcinoma, and its precursor lesion Barrett's esophagus, compared with esophageal squamous cell carcinoma. We propose that although great strides have been made, certain questions remain to which answers hopefully will emerge to have an impact on biomarker diagnostics and translational therapeutics.

Engineered hydrogel reveals contribution of matrix mechanics to esophageal adenocarcinoma and identifies matrix-activated therapeutic targets.

Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying protumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform for identifying potential therapeutic targets to disrupt the contribution of protumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC and inform the development of therapeutics that target ECM stiffness in EAC.

When You Come to a Fork in the Road, Take It: Wnt Signaling Activates Multiple Pathways through the APC/Axin/GSK-3 Complex.

The Wnt signaling pathway is a highly conserved regulator of metazoan development and stem cell maintenance. Activation of Wnt signaling is an early step in diverse malignancies. Work over the past four decades has defined a "canonical" Wnt pathway that is initiated by Wnt proteins, secreted glycoproteins that bind to a surface receptor complex and activate intracellular signal transduction by inhibiting a catalytic complex composed of the classical tumor suppressor Adenomatous Polyposis Coli (APC), Axin, and Glycogen Synthase Kinase-3 (GSK-3). The best characterized effector of this complex is ß-catenin, which is stabilized by inhibition of GSK-3, allowing ß-catenin entrance to the nucleus and activation of Wnt target gene transcription, leading to multiple cancers when inappropriately activated. However, canonical Wnt signaling through the APC/Axin/GSK-3 complex impinges on other effectors, independently of ß-catenin, including the mechanistic Target of Rapamycin (mTOR), regulators of protein stability, mitotic spindle orientation, and Hippo signaling. This review focuses on these alternative effectors of the canonical Wnt pathway and how they may contribute to cancers.

p53 Gain-of-Function Mutation Induces Metastasis via BRD4-Dependent CSF-1 Expression.

TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC) and other SCCs and are associated with a proclivity for metastasis. Here, we report that colony-stimulating factor-1 (CSF-1) expression is upregulated significantly in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. The p53-R172H-dependent CSF-1 signaling, through its cognate receptor CSF-1R, increases tumor cell invasion and lung metastasis, which in turn is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). In Trp53R172H tumor cells, p53 occupies the Csf-1 promoter. The Csf-1 locus is enriched with histone 3 lysine 27 acetylation (H3K27ac), which is likely permissive for fostering an interaction between bromodomain-containing domain 4 (BRD4) and p53-R172H to regulate Csf-1 transcription. Inhibition of BRD4 not only reduces tumor invasion and lung metastasis but also reduces circulating CSF-1 levels. Overall, our results establish a novel p53-R172H-dependent BRD4-CSF-1 axis that promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult-to-treat disease. SIGNIFICANCE: The invasion-metastasis cascade is a recalcitrant barrier to effective cancer therapy. We establish that the p53-R172H-dependent BRD4-CSF-1 axis is a mediator of prometastatic properties, correlates with patient survival and tumor stages, and its inhibition significantly reduces tumor cell invasion and lung metastasis. This axis can be exploited for therapeutic advantage. This article is featured in Selected Articles from This Issue, p. 2489.

Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy.

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Upregulation of the major p63 isoform ΔNp63α, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

IL-1ß transgenic mouse model of inflammation driven esophageal and oral squamous cell carcinoma.

Chronic inflammation is integral to the development of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), although the latter has not been associated with reflux esophagitis. The L2-IL-1ß transgenic mice, expressing human interleukin (IL)-1ß in the oral, esophageal and forestomach squamous epithelia feature chronic inflammation and a stepwise development of Barrett's esophagus-like metaplasia, dysplasia and adenocarcinoma at the squamo-columnar junction. However, the functional consequences of IL-1ß-mediated chronic inflammation in the oral and esophageal squamous epithelia remain elusive. We report for the first time that in addition to the previously described Barrett's esophagus-like metaplasia, the L2-IL-1ß mice also develop squamous epithelial dysplasia with progression to squamous cell carcinoma (SCC) in the esophagus and the tongue. L2-IL-1ß showed age-dependent progression of squamous dysplasia to SCC with approximately 40% (n = 49) and 23.5% (n = 17) incidence rates for esophageal and tongue invasive SCC respectively, by 12-15 months of age. Interestingly, SCC development and progression in L2-IL-1ß was similar in both Germ Free (GF) and Specific Pathogen Free (SPF) conditions. Immunohistochemistry revealed a T cell predominant inflammatory profile with enhanced expression of Ki67, Sox2 and the DNA double-strand break marker, γ-H2AX, in the dysplastic squamous epithelia of L2-IL-1ß mice. Pro-inflammatory cytokines, immunomodulatory players, chemoattractants for inflammatory cells (T cells, neutrophils, eosinophils, and macrophages) and oxidative damage marker, iNOS, were significantly increased in the esophageal and tongue tissues of L2-IL-1ß mice. Our recent findings have expanded the translational utility of the IL-1ß mouse model to aid in further characterization of the key pathways of inflammation driven BE and EAC as well as ESCC and Oral SCC.

LIN28B promotes cell invasion and colorectal cancer metastasis via CLDN1 and NOTCH3.

The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unraveled a potentially novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2, and LoVo) with either knockdown or overexpression of LIN28B, we identified claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and posttranscriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a potentially novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B/CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by posttranscriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models. Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration. Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals. Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation.

The mitochondrial Ca 2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation.

The mitochondrial uniporter (MCU) Ca 2+ ion channel represents the primary means for Ca 2+ uptake into mitochondria. Here we employed in vitro and in vivo models with MCU genetically eliminated to understand how MCU contributes to tumor formation and progression. Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced mitochondrial Ca 2+ uptake, suppression of inactivating-phosphorylation of pyruvate dehydrogenase, a modest increase of basal mitochondrial respiration and a significant increase of acute Ca 2+ -dependent stimulation of mitochondrial respiration. Inhibition of mitochondrial Ca 2+ uptake by genetic deletion of MCU markedly inhibited growth of HEK293T cells and of transformed fibroblasts in mouse xenograft models. Reduced tumor growth was primarily a result of substantially reduced proliferation and fewer mitotic cells in vivo , and slower cell proliferation in vitro associated with delayed progression through S-phase of the cell cycle. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro , both predictors of metastatic potential. Surprisingly, mitochondrial matrix Ca 2+ concentration, membrane potential, global dehydrogenase activity, respiration and ROS production were unchanged by genetic deletion of MCU in transformed cells. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca 2+ signals. Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on mitochondrial Ca 2+ uptake for cell metabolism and Ca 2+ dynamics necessary for cell-cycle progression and cell proliferation.

Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion.

BACKGROUND & AIMS: Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown. METHODS: Using CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids. Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing. Tumorigenicity and immune evasion of organoids were monitored by allograft transplantation. Human ESCC single-cell RNA sequencing data sets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion. RESULTS: We established 32 genetically engineered esophageal organoids and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple-knockout induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN knockout also generates an immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via nuclear factor-κB. Moreover, comparative single-cell transcriptomic analyses stratified patients with ESCC and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1. CONCLUSIONS: Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable option for targeting PCN-type ESCC.

Tumor-Derived CCL5 Recruits Cancer-Associated Fibroblasts and Promotes Tumor Cell Proliferation in Esophageal Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) can promote tumor growth, metastasis, and therapeutic resistance in esophageal squamous cell carcinoma (ESCC), but the mechanisms of action remain elusive. Our objective was to identify secreted factor(s) that mediate the communication between CAFs and ESCC tumor cells with the aim of identifying potential druggable targets. Through unbiased cytokine arrays, we have identified CC motif chemokine ligand 5 (CCL5) as a secreted factor that is increased upon co-culture of ESCC cells and CAFs, which we replicated in esophageal adenocarcinoma (EAC) with CAFs. Loss of tumor-cell-derived CCL5 reduces ESCC cell proliferation in vitro and in vivo and we propose this is mediated, in part, by a reduction in ERK1/2 signaling. Loss of tumor-derived CCL5 reduces the percentage of CAFs recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists, namely Maraviroc. Maraviroc treatment reduced tumor volume, CAF recruitment, and ERK1/2 signaling in vivo, thus, mimicking the effects observed with genetic loss of CCL5. High CCL5 or CCR5 expression is associated with worse prognosis in low-grade esophageal carcinomas. IMPLICATIONS: These data highlight the role of CCL5 in tumorigenesis and the therapeutic potential of targeting the CCL5-CCR5 axis in ESCC.

Specific regulation of BACH1 by the hotspot mutant p53R175H reveals a distinct gain-of-function mechanism.

Although the gain of function (GOF) of p53 mutants is well recognized, it remains unclear whether different p53 mutants share the same cofactors to induce GOFs. In a proteomic screen, we identified BACH1 as a cellular factor that recognizes the p53 DNA-binding domain depending on its mutation status. BACH1 strongly interacts with p53R175H but fails to effectively bind wild-type p53 or other hotspot mutants in vivo for functional regulation. Notably, p53R175H acts as a repressor for ferroptosis by abrogating BACH1-mediated downregulation of SLC7A11 to enhance tumor growth; conversely, p53R175H promotes BACH1-dependent tumor metastasis by upregulating expression of pro-metastatic targets. Mechanistically, p53R175H-mediated bidirectional regulation of BACH1 function is dependent on its ability to recruit the histone demethylase LSD2 to target promoters and differentially modulate transcription. These data demonstrate that BACH1 acts as a unique partner for p53R175H in executing its specific GOFs and suggest that different p53 mutants induce their GOFs through distinct mechanisms.

Phase Ib and Expansion Study of Gemcitabine, Nab-Paclitaxel, and Ficlatuzumab in Patients With Metastatic Pancreatic Cancer.

BACKGROUND: In preclinical pancreatic ductal adenocarcinoma (PDAC) models, inhibition of hepatocyte growth factor (HGF) signaling using ficlatuzumab, a recombinant humanized anti-HGF antibody, and gemcitabine reduced tumor burden. METHODS: Patients with previously untreated metastatic PDAC enrolled in a phase Ib dose escalation study with 3 + 3 design of 2 dose cohorts of ficlatuzumab 10 and 20 mg/kg administered intravenously every other week with gemcitabine 1000 mg/m2 and albumin-bound paclitaxel 125 mg/m2 given 3 weeks on and 1 week off. This was followed by an expansion phase at the maximally tolerated dose of the combination. RESULTS: Twenty-six patients (sex, 12 male:14 female; median age, 68 years [range, 49-83 years]) were enrolled, 22 patients were evaluable. No dose-limiting toxicities were identified (N = 7 pts) and ficlatuzumab at 20 mg/kg was chosen as the maximum tolerated dose. Among the 21 patients treated at the MTD, best response by RECISTv1.1: 6 (29%) partial response, 12 (57%) stable disease, 1 (5%) progressive disease, and 2 (9%) not evaluable. Median progression-free survival and overall survival times were 11.0 months (95% CI, 7.6-11.4 months) and 16.2 months (95% CI, 9.1 months to not reached), respectively. Toxicities attributed to ficlatuzumab included hypoalbuminemia (grade 3, 16%; any grade, 52%) and edema (grade 3, 8%; any grade, 48%). Immunohistochemistry for c-Met pathway activation demonstrated higher tumor cell p-Met levels in patients who experienced response to therapy. CONCLUSION: In this phase Ib trial, ficlatuzumab, gemcitabine, and albumin-bound paclitaxel were associated with durable treatment responses and increased rates of hypoalbuminemia and edema.

Tumor Niche Network-Defined Subtypes Predict Immunotherapy Response of Esophageal Squamous Cell Cancer.

Despite the promising outcomes of immune checkpoint blockade (ICB), resistance to ICB presents a new challenge. Therefore, selecting patients for specific ICB applications is crucial for maximizing therapeutic efficacy. Herein we curated 69 human esophageal squamous cell cancer (ESCC) patients' tumor microenvironment (TME) single-cell transcriptomic datasets to subtype ESCC. Integrative analyses of the cellular network transcriptional signatures of T cells, myeloid cells, and fibroblasts define distinct ESCC subtypes characterized by T cell exhaustion, Interferon (IFN) a/b signaling, TIGIT enrichment, and specific marker genes. Furthermore, this approach classifies ESCC patients into ICB responders and non-responders, as validated by liquid biopsy single-cell transcriptomics. Our study stratifies ESCC patients based on TME transcriptional network, providing novel insights into tumor niche remodeling and predicting ICB responses in ESCC patients.

Metastatic colorectal cancer: mechanisms and emerging therapeutics.

Metastatic colorectal cancer (mCRC) remains a lethal disease with an approximately 14% 5-year survival rate. While early-stage colorectal cancer (CRC) can be cured by surgery with or without adjuvant chemotherapy, mCRC cannot be eradicated due to a large burden of disseminated cancer cells comprising therapy-resistant metastasis-competent cells. To address this gap, recent studies have focused on further elucidating the molecular mechanisms underlying colorectal metastasis and recognizing the limitations of available therapeutic interventions. In this review, we discuss newfound factors that regulate CRC cell dissemination and colonization of distant organs, such as genetic mutations, identification of metastasis-initiating cells (MICs), epithelial-mesenchymal transition (EMT), and the tumor microenvironment (TME). We also review current treatments for mCRC, therapeutic regimens undergoing clinical trials, and trending preclinical studies being investigated to target treatment-resistant mCRC.

SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division.

BACKGROUND & AIMS: Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. METHODS: Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. RESULTS: Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. CONCLUSION: SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.

KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition.

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

Tumor-Suppressive and Immune-Stimulating Roles of Cholesterol 25-hydroxylase in Pancreatic Cancer Cells.

Cholesterol dependence is an essential characteristic of pancreatic ductal adenocarcinoma (PDAC). Cholesterol 25-hydroxylase (CH25H) catalyzes monooxygenation of cholesterol into 25-hydroxycholesterol, which is implicated in inhibiting cholesterol biosynthesis and in cholesterol depletion. Here, we show that, within PDAC cells, accumulation of cholesterol was facilitated by the loss of CH25H. Methylation of the CH25H gene and decreased levels of CH25H expression occurred in human pancreatic cancers and was associated with poor prognosis. Knockout of Ch25h in mice accelerated progression of Kras-driven pancreatic intraepithelial neoplasia. Conversely, restoration of CH25H expression in human and mouse PDAC cells decreased their viability under conditions of cholesterol deficit, and decelerated tumor growth in immune competent hosts. Mechanistically, the loss of CH25H promoted autophagy resulting in downregulation of MHC-I and decreased CD8+ T-cell tumor infiltration. Re-expression of CH25H in PDAC cells combined with immune checkpoint inhibitors notably inhibited tumor growth. We discuss additional benefits that PDAC cells might gain from inactivation of CH25H and the potential translational importance of these findings for therapeutic approaches to PDAC. IMPLICATIONS: Loss of CH25H by pancreatic cancer cells may stimulate tumor progression and interfere with immunotherapies.

Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.