Optimization of a mouse model of pancreatic cancer to simulate the human phenotypes of metastasis and cachexia.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging. METHODS: Here, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions. RESULTS: These modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis. CONCLUSION: Together, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics.

High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells.

Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

MYC is sufficient to generate mid-life high-grade serous ovarian and uterine serous carcinomas in a p53-R270H mouse model.

Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment is understood. However, previous GEMMs of high-grade serous ovarian cancer (HGSOC) have had to utilize genetics rarely or never found in human HGSOC to yield ovarian cancer within the lifespan of a mouse. MYC, an oncogene, is amongst the most amplified genes in HGSOC, but it has not previously been utilized to drive HGSOC GEMMs. We coupled Myc and dominant negative mutant p53-R270H with a fallopian tube epithelium-specific promoter Ovgp1 to generate a new GEMM of HGSOC. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium (FTE). Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Taken together, these results demonstrate the Myc and Trp53-R270H transgene was able to recapitulate many phenotypic hallmarks of HGSOC through the utilization of strictly human-mimetic genetic hallmarks of HGSOC. This new mouse model enables further exploration of ovarian cancer pathogenesis, particularly in the 50% of HGSOC which lack homology directed repair mutations. Histological and transcriptomic findings are consistent with the hypothesis that uterine serous cancer may originate from the fallopian tube epithelium.

Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

Cellular and Molecular Mechanisms of MEK1 Inhibitor-Induced Cardiotoxicity.

Background: Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized. Objectives: The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function. Methods: Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery. Results: Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment. Conclusions: These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention.

Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism.

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language, and cognitive deficits. A mouse model of this syndromic ASD (Mef2c-Het) recapitulates many of the MEF2C haploinsufficiency syndrome-linked behaviors, including communication deficits. We show here that Mef2c-Het mice of both sexes exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types; and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments.SIGNIFICANCE STATEMENT This is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD-related behaviors, including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, subcellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.

Aging-dependent mitochondrial dysfunction mediated by ceramide signaling inhibits antitumor T cell response.

We lack a mechanistic understanding of aging-mediated changes in mitochondrial bioenergetics and lipid metabolism that affect T cell function. The bioactive sphingolipid ceramide, induced by aging stress, mediates mitophagy and cell death; however, the aging-related roles of ceramide metabolism in regulating T cell function remain unknown. Here, we show that activated T cells isolated from aging mice have elevated C14/C16 ceramide accumulation in mitochondria, generated by ceramide synthase 6, leading to mitophagy/mitochondrial dysfunction. Mechanistically, aging-dependent mitochondrial ceramide inhibits protein kinase A, leading to mitophagy in activated T cells. This aging/ceramide-dependent mitophagy attenuates the antitumor functions of T cells in vitro and in vivo. Also, inhibition of ceramide metabolism or PKA activation by genetic and pharmacologic means prevents mitophagy and restores the central memory phenotype in aging T cells. Thus, these studies help explain the mechanisms behind aging-related dysregulation of T cells' antitumor activity, which can be restored by inhibiting ceramide-dependent mitophagy.

Pro-Survival Lipid Sphingosine-1-Phosphate Metabolically Programs T Cells to Limit Anti-tumor Activity.

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.

Complement C3a and C5a receptors promote GVHD by suppressing mitophagy in recipient dendritic cells.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT). DCs play critical roles in GVHD induction. Modulating autophagy represents a promising therapeutic strategy for the treatment of immunological diseases. Complement receptors C3aR/C5aR expressed on DCs regulate immune responses by translating extracellular signals into intracellular activity. In the current study, we found that C3aR/C5aR deficiency enhanced ceramide-dependent lethal mitophagy (CDLM) in DCs. Cotransfer of host-type C3aR-/-/C5aR-/- DCs in the recipients significantly improved GVHD outcome after allogeneic HCT, primarily through enhancing CDLM in DCs. C3aR/C5aR deficiency in the host hematopoietic compartment significantly reduced GVHD severity via impairing Th1 differentiation and donor T cell glycolytic activity while enhancing Treg generation. Prophylactic treatment with C3aR/C5aR antagonists effectively alleviated GVHD while maintaining the graft-versus-leukemia (GVL) effect. Altogether, we demonstrate that inhibiting C3aR/C5aR induces lethal mitophagy in DCs, which represents a potential therapeutic approach to control GVHD while preserving the GVL effect.

HPV/E7 induces chemotherapy-mediated tumor suppression by ceramide-dependent mitophagy.

Human papillomavirus (HPV) infection is linked to improved survival in response to chemo-radiotherapy for patients with oropharynx head and neck squamous cell carcinoma (HNSCC). However, mechanisms involved in increased HNSCC cell death by HPV signaling in response to therapy are largely unknown. Here, using molecular, pharmacologic and genetic tools, we show that HPV early protein 7 (E7) enhances ceramide-mediated lethal mitophagy in response to chemotherapy-induced cellular stress in HPV-positive HNSCC cells by selectively targeting retinoblastoma protein (RB). Inhibition of RB by HPV-E7 relieves E2F5, which then associates with DRP1, providing a scaffolding platform for Drp1 activation and mitochondrial translocation, leading to mitochondrial fission and increased lethal mitophagy. Ectopic expression of a constitutively active mutant RB, which is not inhibited by HPV-E7, attenuated ceramide-dependent mitophagy and cell death in HPV(+) HNSCC cells. Moreover, mutation of E2F5 to prevent Drp1 activation inhibited mitophagy in HPV(+) cells. Activation of Drp1 with E2F5-mimetic peptide for inducing Drp1 mitochondrial localization enhanced ceramide-mediated mitophagy and led to tumor suppression in HPV-negative HNSCC-derived xenograft tumors in response to cisplatin in SCID mice.

Slug expression inhibits calcitriol-mediated sensitivity to radiation in colorectal cancer.

Recently, a reciprocal relationship between calcitriol and epithelial-to-mesenchymal transition has been described. Therefore, we hypothesized that calcitriol (1α,25-dihydroxyvitamin D3) would enhance radiation sensitivity in colorectal cancer regulated by epithelial mesenchymal transition. Vitamin-D receptor, E-cadherin and vimentin protein as well as E-cadherin, Snail and Slug mRNA levels were assessed in a panel of human colorectal cancer cell lines at baseline and in response calcitriol. We defined cell lines as calcitriol sensitive based on demonstrating an enhanced epithelial phenotype with increased E-cadherin, reduced vimentin and decreased expression of Snail and Slug as well as decreased cellular migration in response to calcitriol. In calcitriol sensitive cells, including DLD-1 and HCT116, 24 h calcitriol pre-treatment enhanced the radiation sensitivity by 2.3- and 2.6-fold, respectively, at 4 Gy (P < 0.05). In contrast, SW620 cells with high baseline mesenchymal features including high Slug and vimentin expression with low E-cadherin expression demonstrated no significant radiation sensitizing response to calcitriol treatment. Similarly, transfection of Slug in the calcitriol sensitive colon cancer cell lines, DLD-1 and HCT 116, completely inhibited the radiation sensitizing effect of calcitriol. Collectively, we demonstrate that calcitriol can enhance the therapeutic effects of radiation in colon cancer cells and Slug expression mitigates this observed effect potentially representing an effective biomarker for calcitriol therapy.

Slug expression enhances tumor formation in a noninvasive rectal cancer model.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a series of molecular changes allowing epithelial cancer cells to acquire properties of mesenchymal cells: increased motility, invasion, and protection from apoptosis. Transcriptional regulators such as Slug mediate EMT, working in part to repress E-cadherin transcription. We report a novel, noninvasive in vivo rectal cancer model to explore the role of Slug in colorectal cancer (CRC) tumor development. METHODS: For the generation of DLD-1 cells overexpressing Slug (Slug DLD-1), a Slug or empty (Empty DLD-1) pCMV-3Tag-1 (kanamycin-resistant) vector was used for transfection. Cells were evaluated for Slug and E-cadherin expression, and cell migration and invasion. For the in vivo study, colon cancer cells (parental DLD-1, Slug DLD-1, empty DLD-1, and HCT-116) were submucosally injected into the posterior rectum of nude mice using endoscopic guidance. After 28 d, tumors were harvested and tissue was analyzed. RESULTS: Slug expression in our panel of colon cancer cell lines was inversely correlated with E-cadherin expression and enhanced migration/invasion. Slug DLD-1 cells demonstrated a 21-fold increased Slug and 19-fold decreased E-cadherin expression compared with empty DLD-1. Similarly, the Slug DLD-1 cells had significantly enhanced cellular migration and invasion. In the orthotopic rectal cancer model, Slug DLD-1 cells formed rectal tumors in 9/10 (90%) of the mice (mean volume = 458 mm(3)) compared with only 1/10 (10%) with empty DLD-1 cells. CONCLUSION: Slug mediates EMT with enhanced in vivo rectal tumor formation. Our noninvasive in vivo model enables researchers to explore the molecular consequences of altered genes in a clinically relevant rectal cancer in an effort to develop novel therapeutic approaches for patients with rectal cancer.