The Spatial Extracellular Proteomic Tumor Microenvironment Distinguishes Molecular Subtypes of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidin-domain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.

Dishevelled 2 regulates cancer cell proliferation and T cell mediated immunity in HER2-positive breast cancer.

BACKGROUND: Dishevelled paralogs (DVL1, 2, 3) are key mediators of Wnt pathway playing a role in constitutive oncogenic signaling influencing the tumor microenvironment. While previous studies showed correlation of ß-catenin with T cell gene expression, little is known about the role of DVL2 in modulating tumor immunity. This study aimed to uncover the novel interaction between DVL2 and HER2-positive (HER2+) breast cancer (BC) in regulating tumor immunity and disease progression. METHODS: DVL2 loss of function studies were performed with or without a clinically approved HER2 inhibitor, Neratinib in two different HER2+ BC cell lines. We analyzed RNA (RT-qPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. A pilot study in 24 HER2+ BC patients was performed to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p < 0.05. RESULTS: DVL2 regulates the transcription of immune modulatory genes involved in antigen presentation and T cell maintenance. DVL2 loss of function down regulated mRNA expression of Wnt target genes involved in cell proliferation, migration, invasion in HER2+ BC cell lines (±Neratinib). Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown (±Neratinib) resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control in one of the two cell lines used. Analyses on patient tissues who received neoadjuvant chemotherapy (n = 14) further demonstrate that higher DVL2 expression at baseline biopsy pose a significant negative correlation with % CD8α levels (r = - 0.67, p < 0.05) while have a positive correlation with NLR (r = 0.58, p < 0.05), where high NLR denotes worse cancer prognosis. These results from our pilot study reveal interesting roles of DVL2 proteins in regulating tumor immune microenvironment and clinical predictors of survival in HER2+ BC. CONCLUSION: Our study demonstrates potential immune regulatory role of DVL2 proteins in HER2+ BC. More in-depth mechanistic studies of DVL paralogs and their influence on anti-tumor immunity may provide insight into DVLs as potential therapeutic targets benefiting BC patients.

Hepatocellular Carcinoma Chemoprevention with Generic Agents.

Liver cancer, mainly hepatocellular carcinoma (HCC), remains a major cause of cancer-related death worldwide. With the global epidemic of obesity, the major HCC etiologies have been dynamically shifting from viral to metabolic liver diseases. This change has made HCC prevention difficult with increasingly elusive at-risk populations as rational target for preventive interventions. Besides ongoing efforts to reduce obesity and metabolic disorders, chemoprevention in patients who already have metabolic liver diseases may have a significant impact on the poor HCC prognosis. Hepatitis B- and hepatitis C-related HCC incidences have been substantially reduced by the new antivirals, but HCC risk can persist over a decade even after successful viral treatment, highlighting the need for HCC-preventive measures also in these patients. Experimental and retrospective studies have suggested potential utility of generic agents such as lipophilic statins and aspirin for HCC chemoprevention given their well-characterized safety profile, although anticipated efficacy may be modest. In this review, we overview recent clinical and translational studies of generic agents in the context of HCC chemoprevention under the contemporary HCC etiologies. We also discuss newly emerging approaches to overcome the challenges in clinical testing of the agents to facilitate their clinical translation.

Mice with humanized immune system as novel models to study HIV-associated pulmonary hypertension.

People living with HIV and who receive antiretroviral therapy have a significantly improved lifespan, compared to the early days without therapy. Unfortunately, persisting viral replication in the lungs sustains chronic inflammation, which may cause pulmonary vascular dysfunction and ultimate life-threatening Pulmonary Hypertension (PH). The mechanisms involved in the progression of HIV and PH remain unclear. The study of HIV-PH is limited due to the lack of tractable animal models that recapitulate infection and pathobiological aspects of PH. On one hand, mice with humanized immune systems (hu-mice) are highly relevant to HIV research but their suitability for HIV-PH research deserves investigation. On another hand, the Hypoxia-Sugen is a well-established model for experimental PH that combines hypoxia with the VEGF antagonist SU5416. To test the suitability of hu-mice, we combined HIV with either SU5416 or hypoxia. Using right heart catheterization, we found that combining HIV+SU5416 exacerbated PH. HIV infection increases human pro-inflammatory cytokines in the lungs, compared to uninfected mice. Histopathological examinations showed pulmonary vascular inflammation with arterial muscularization in HIV-PH. We also found an increase in endothelial-monocyte activating polypeptide II (EMAP II) when combining HIV+SU5416. Therefore, combinations of HIV with SU5416 or hypoxia recapitulate PH in hu-mice, creating well-suited models for infectious mechanistic pulmonary vascular research in small animals.

Hepatocellular carcinoma chemoprevention by targeting the angiotensin-converting enzyme and EGFR transactivation.

Hepatocellular carcinoma (HCC) is a leading cause of death among cirrhotic patients, for which chemopreventive strategies are lacking. Recently, we developed a simple human cell-based system modeling a clinical prognostic liver signature (PLS) predicting liver disease progression and HCC risk. In a previous study, we applied our cell-based system for drug discovery and identified captopril, an approved angiotensin converting enzyme (ACE) inhibitor, as a candidate compound for HCC chemoprevention. Here, we explored ACE as a therapeutic target for HCC chemoprevention. Captopril reduced liver fibrosis and effectively prevented liver disease progression toward HCC development in a diethylnitrosamine (DEN) rat cirrhosis model and a diet-based rat model for nonalcoholic steatohepatitis-induced (NASH-induced) hepatocarcinogenesis. RNA-Seq analysis of cirrhotic rat liver tissues uncovered that captopril suppressed the expression of pathways mediating fibrogenesis, inflammation, and carcinogenesis, including epidermal growth factor receptor (EGFR) signaling. Mechanistic data in liver disease models uncovered a cross-activation of the EGFR pathway by angiotensin. Corroborating the clinical translatability of the approach, captopril significantly reversed the HCC high-risk status of the PLS in liver tissues of patients with advanced fibrosis. Captopril effectively prevents fibrotic liver disease progression toward HCC development in preclinical models and is a generic and safe candidate drug for HCC chemoprevention.

Tumor-Infiltrating Lymphocytes (TILs) as a Biomarker of Abscopal Effect of Cryoablation in Breast Cancer: A Pilot Study.

BACKGROUND: Morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer is gaining momentum as an immunological biomarker. This experiment evaluates the role of TILs in distant tumors as a measure of abscopal effect from cryoablation of breast cancer. METHODS: BALB/c mice underwent bilateral orthotopic transplant with 4T1-12B (triple-negative) cells. At 2 weeks, left tumors were treated by either resection (standard of care group) or cryoablation (intervention group) followed by resection of the distant right tumors 1 week posttreatment. TIL scores were calculated from hematoxylin and eosin-stained sections and phenotyped for cytotoxic T-lymphocyte (CTL) markers by immunofluorescence. Primarily resected tumors served as baseline (Tbaseline), whereas resected distant right-sided served as the readout for abscopal effect (AbsRes or AbsCryo). Mice were monitored for tumor recurrence and metastasis. RESULTS: The AbsCryo had a significant mean (SD) increase in stromal (2.8 [1.1]%; p = 0.015) and invasive margin TILs (50 [12]%; p = 0.02) compared with TBaseline (1.0 [0]% and 31 [4.9]%, respectively). CTL phenotyping revealed a significant increase in mean (SD) CD8+ T cells (15.7 [12.1]; p = 0.02) and granzyme B (4.8 [3.6]; p = 0.048) for the AbsCryo compared with TBaseline (5.2 [4.7] and 2.4 [0.9], respectively). Posttreatment, the cryoablation group had no recurrence or metastasis, whereas the resected group showed local recurrence and lung metastasis in 40% of the mice. Postprocedure increase in TIL score of distant tumors was associated with decrease in tumor relapse (p = 0.02). CONCLUSIONS: Cryoablation induced a robust tumor-specific TIL response compared with resection, suggesting an abscopal effect leading to the prevention of cancer recurrence and metastasis.

Dishevelled-1 DIX and PDZ domain lysine residues regulate oncogenic Wnt signaling.

DVL proteins are central mediators of the Wnt pathway and relay complex input signals into different branches of the Wnt signaling network. However, molecular mechanism(s) that regulate DVL-mediated relay of Wnt signals still remains unclear. Here, for the first time, we elucidate the functional significance of three DVL-1 lysines (K/Lys) which are subject to post-translational acetylation. We demonstrate that K34 Lys residue in the DIX domain regulates subcellular localization of ß-catenin, thereby influencing downstream Wnt target gene expression. Additionally, we show that K69 (DIX domain) and K285 (PDZ domain) regulate binding of DVL-1 to Wnt target gene promoters and modulate expression of Wnt target genes including CMYC, OCT4, NANOG, and CCND1, in cell line models and xenograft tumors. Finally, we report that conserved DVL-1 lysines modulate various oncogenic functions such as cell migration, proliferation, cell-cycle progression, 3D-spheroid formation and in-vivo tumor growth in breast cancer models. Collectively, these findings highlight the importance of DVL-1 domain-specific lysines which were recently shown to be acetylated and characterize their influence on Wnt signaling. These site-specific modifications may be subject to regulation by therapeutics already in clinical use (lysine deacetylase inhibitors such as Panobinostat and Vorinostat) or may possibly have prognostic utility in translational efforts that seek to modulate dysfunctional Wnt signaling.

Mechanisms of endocrine therapy resistance in breast cancer.

The most commonly diagnosed breast cancer (BC) subtype is characterized by estrogen receptor (ER) expression. Treatment of this BC subtype typically involves modalities that either suppress the production of estrogen or impede the binding of estrgen to its receptors, constituting the basis for endocrine therapy. While many patients have benefitted from endocrine therapy with clear reduction in mortality and cancer recurrence, one of the clinical hurdles that remain involves overcoming intrinsic (de novo) or acquired resistance to endocrine therapy driven by diverse and complex changes occurring in the tumor microenvironment. Moreover, such resistance may persist even after progression through additional antiestrogen therapies thus demonstrating the importance of further investigation of mechanisms of ER modulation. Here, we discuss a number of advances that provide a better understanding of the complex mechanistic basis for resistance to endocrine therapy as well as future therapeutic maneuvers that may break this resistance.

Regulation of aromatase in cancer.

The regulation of aromatase, an enzyme involved in the biosynthesis of estrogen in normal and cancer cells, has been associated with growth factor signaling and immune response modulation. The tissue-specific regulatory roles of these factors are of particular importance as local aromatase expression is strongly linked to cancer development/progression and disease outcomes in patients. Therefore, aromatase has become a chemotherapeutic target and aromatase inhibitors (AIs) are used in the clinic for treating hormone-dependent cancers. Although AIs have shown promising results in the treatment of cancers, the emerging increase in AI-resistance necessitates the development of new and improved targeted therapies. This review discusses the role of tumor and stromal-derived growth factors and immune cell modulators in regulating aromatase. Current single-agent and combination therapies with or without AIs targeting growth factors and immune checkpoints are also discussed. This review highlights recent studies that show new connections between growth factors, mediators of immune response, and aromatase regulation.

The Versatility of Sirtuin-1 in Endocrinology and Immunology.

Sirtuins belong to the class III family of NAD-dependent histone deacetylases (HDAC) and are involved in diverse physiological processes that range from regulation of metabolism and endocrine function to coordination of immunity and cellular responses to stress. Sirtuin-1 (SIRT1) is the most well-studied family member and has been shown to be critically involved in epigenetics, immunology, and endocrinology. The versatile roles of SIRT1 include regulation of energy sensing metabolic homeostasis, deacetylation of histone and non-histone proteins in numerous tissues, neuro-endocrine regulation via stimulation of hypothalamus-pituitary axes, synthesis and maintenance of reproductive hormones via steroidogenesis, maintenance of innate and adaptive immune system via regulation of T- and B-cell maturation, chronic inflammation and autoimmune diseases. Moreover, SIRT1 is an appealing target in various disease contexts due to the promise of pharmacological and/or natural modulators of SIRT1 activity within the context of endocrine and immune-related disease models. In this review we aim to provide a broad overview on the role of SIRT1 particularly within the context of endocrinology and immunology.

Identification of Novel MeCP2 Cancer-Associated Target Genes and Post-Translational Modifications.

Abnormal regulation of DNA methylation and its readers has been associated with a wide range of cellular dysfunction. Disruption of the normal function of DNA methylation readers contributes to cancer progression, neurodevelopmental disorders, autoimmune disease and other pathologies. One reader of DNA methylation known to be especially important is MeCP2. It acts a bridge and connects DNA methylation with histone modifications and regulates many gene targets contributing to various diseases; however, much remains unknown about how it contributes to cancer malignancy. We and others previously described novel MeCP2 post-translational regulation. We set out to test the hypothesis that MeCP2 would regulate novel genes linked with tumorigenesis and that MeCP2 is subject to additional post-translational regulation not previously identified. Herein we report novel genes bound and regulated by MeCP2 through MeCP2 ChIP-seq and RNA-seq analyses in two breast cancer cell lines representing different breast cancer subtypes. Through genomics analyses, we localize MeCP2 to novel gene targets and further define the full range of gene targets within breast cancer cell lines. We also further examine the scope of clinical and pre-clinical lysine deacetylase inhibitors (KDACi) that regulate MeCP2 post-translationally. Through proteomics analyses, we identify many additional novel acetylation sites, nine of which are mutated in Rett Syndrome. Our study provides important new insight into downstream targets of MeCP2 and provide the first comprehensive map of novel sites of acetylation associated with both pre-clinical and FDA-approved KDACi used in the clinic. This report examines a critical reader of DNA methylation and has important implications for understanding MeCP2 regulation in cancer models and identifying novel molecular targets associated with epigenetic therapies.

Renin angiotensin system inhibition attenuates adipocyte-breast cancer cell interactions.

Obesity is a significant breast cancer (BC) risk factor and is associated with 20-40% increased risk in obese post-menopausal women compared to their lean counterparts. Several obesity-related metabolic dysregulations have been linked to BC risk, including overactivation of the renin-angiotensin system (RAS). Currently, RAS inhibitors including angiotensin converting enzyme inhibitor (ACEi) and AT1 receptor blockers (ARBs), are used as safe and effective anti-hypertensive therapies in BC patients. However, it is uncertain how inhibition of RAS in adipose tissue impacts obesity-BC crosstalk. We hypothesized that adipose RAS inhibition will reduce BC cell motility and inflammation. We determined (1) the direct effects of Ang II, ACEi (captopril; Cap) or ARB (telmisartan; Tel) on receptor positive MCF-7 and receptor triple negative MDA-MB-231 cells; and (2) the effects of conditioned media (CM) from human mesenchymal stem cells differentiated into adipocytes, which were pretreated with RAS inhibitors, on BC cells. We demonstrated that direct treatments of BC cells with Ang II, Cap or Tel did not alter inflammatory cytokines in either BC cell line. However, CM from Ang II-pretreated adipocytes significantly increased secretion of pro-inflammatory markers at protein level. RAS inhibitors reduced their secretion in MDA-MB-231, but not in MCF-7 cells. Additionally, CM from adipocytes treated with RAS inhibitors significantly reduced markers of inflammation, fat synthesis, and angiogenesis in both BC cell lines. Furthermore, CM from ACEi pretreated adipocytes reduced cell motility in both BC cell lines. Findings from our study indicate an important role of adipose RAS inhibition in adipocyte and BC cell crosstalk.

Autophagy in metabolic syndrome: breaking the wheel by targeting the renin-angiotensin system.

Metabolic syndrome (MetS) is a complex, emerging epidemic which disrupts the metabolic homeostasis of several organs, including liver, heart, pancreas, and adipose tissue. While studies have been conducted in these research areas, the pathogenesis and mechanisms of MetS remain debatable. Lines of evidence show that physiological systems, such as the renin-angiotensin system (RAS) and autophagy play vital regulatory roles in MetS. RAS is a pivotal system known for controlling blood pressure and fluid balance, whereas autophagy is involved in the degradation and recycling of cellular components, including proteins. Although RAS is activated in MetS, the interrelationship between RAS and autophagy varies in glucose homeostatic organs and their cross talk is poorly understood. Interestingly, autophagy is attenuated in the liver during MetS, whereas autophagic activity is induced in adipose tissue during MetS, indicating tissue-specific discordant roles. We discuss in vivo and in vitro studies conducted in metabolic tissues and dissect their tissue-specific effects. Moreover, our review will focus on the molecular mechanisms by which autophagy orchestrates MetS and the ways future treatments could target RAS in order to achieve metabolic homeostasis.

Combined Effects of Eicosapentaenoic Acid and Adipocyte Renin-Angiotensin System Inhibition on Breast Cancer Cell Inflammation and Migration.

Obesity is a major risk factor for breast cancer (BC). Obesity-related metabolic alterations such as inflammation and overactivation of the adipose renin-angiotensin system (RAS) may contribute to the progression of BC. Clinically used antihypertensive drugs such as angiotensin-converting enzyme inhibitors (ACE-I) and dietary bioactive components such as eicosapentaenoic acid (EPA) are known for their anti-inflammatory and adipose RAS blocking properties. However, whether EPA enhances the protective effects of ACE-I in lessening adipocyte inflammation on BC cells has not been studied. We hypothesized that combined EPA and ACE-I would attenuate BC cell inflammation and migration possibly via adipose RAS inhibition. To test our hypothesis, we examined the (i) direct effects of an ACE-I (captopril (CAP)) or EPA, individually and combined, on MCF-7 and MDA-MB-231 human BC cells, and the (ii) effects of conditioned medium (CM) from human adipocytes pretreated with the abovementioned agents on BC cells. We demonstrated that CM from adipocytes pretreated with EPA with or without captopril (but not direct treatments of BC cells) significantly reduced proinflammatory cytokines expression in both BC cell lines. Additionally, cell migration was reduced in MDA-MB-231 cells in response to both direct and CM-mediated CAP and/or EPA treatments. In summary, our study provides a significant insight into added benefits of combining anti-inflammatory EPA and antihypertensive ACE-I to attenuate the effects of adipocytes on breast cancer cell migration and inflammation.

Protective effects of eicosapentaenoic acid in adipocyte-breast cancer cell cross talk.

Breast cancer is the leading cause of death in women among all cancer types. Obesity is one of the factors that promote progression of breast cancer, especially in post-menopausal women. Increasingly, adipose tissue is recognized for its active role in the tumor microenvironment. We hypothesized that adipocytes conditioned medium can impact breast cancer progression by increasing inflammatory cytokines production by cancer cells, and subsequently increasing their motility. By contrast, eicosapentaenoic acid (EPA), an anti-inflammatory n-3 polyunsaturated fatty acid, reduces adipocyte-secreted inflammatory factors, leading to reduced cancer cell motility. To test these hypotheses, we investigated the direct effects of EPA on MCF-7 and MDA-MB-231 breast cancer cells and the effects of conditioned medium from 3 T3-L1 or human mesenchymal stem cells (HMSC)-derived adipocytes treated with or without EPA supplementation on breast cancer cells. We observed that conditioned medium from HMSC-derived adipocytes significantly increased mRNA transcription levels of cancer-associated genes such as FASN, STAT3 and cIAP2, while EPA-treated HMSC-derived adipocytes significantly reduced mRNA levels of these genes. However, direct EPA treatment significantly reduced mRNA content of these tumor-associated markers (FASN, STAT3, cIAP-2) only in MDA-MB-231 cells not in MCF-7 cells. Conditioned medium from EPA-treated 3 T3-L1 adipocytes further decreased inflammation, cell motility and glycolysis in cancer cells. Our data confirms that adipocytes play a significant role in promoting breast cancer progression and demonstrates that EPA-treated adipocytes reduced the negative impact of adipocyte-secreted factors on breast cancer cell inflammation and migration.

Mechanisms linking the renin-angiotensin system, obesity, and breast cancer.

Obesity is a complex disease and a global epidemic. It is a risk factor for other chronic diseases including breast cancer, especially in women after menopause. Diverse etiologies underlie the relationship between obesity and breast cancer. Adipose tissue is in part responsible for these interactions. In obesity, adipose tissue undergoes several metabolic dysregulations resulting in the secretion of many pro-inflammatory cytokines, growth factors, and hormones which in turn, can promote tumor microenvironment (TME) formation and cancer progression within the breast tissue. Angiotensin II (Ang II) is a well-known hypertensive hormone produced systemically and locally by the renin-angiotensin system (RAS). Activation of this system in obesity is a potential contributor to local and systemic inflammation in breast adipose tissue. Ang II actions are primarily mediated through binding to its two receptors, type 1 (AT1R) and type 2 (AT2R). RAS inhibitors include angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARBs) which are currently prescribed as safe antihypertensive therapies. Recent studies have explored the potential use of ACE-I and ARBs in breast cancer patients as anti-tumor agents. Therefore, it is vital to understand the role of RAS in breast cancer and identify mechanisms of Ang II and RAS inhibitors in the TME and in obesity and breast cancer crosstalk. In this review, we performed a detailed analysis and discussed mechanisms of Ang II-AT1R interactions in breast cancer with emphasis on obesity-associated breast cancer. We further summarized recent in vitro, in vivo and human studies that used ACE-I/ARB interventions to improve breast cancer outcomes.