Oncogene-induced matrix reorganization controls CD8+ T cell function in the soft-tissue sarcoma microenvironment.

CD8+ T cell dysfunction impedes anti-tumor immunity in solid cancers but the underlying mechanisms are diverse and poorly understood. Extracellular matrix (ECM) composition has been linked to impaired T cell migration and enhanced tumor progression; however, impacts of individual ECM molecules on T cell function in the tumor microenvironment (TME) are only beginning to be elucidated. Upstream regulators of aberrant ECM deposition and organization in solid tumors are equally ill-defined. Therefore, we investigated how ECM composition modulates CD8+ T cell function in undifferentiated pleomorphic sarcoma (UPS), an immunologically active desmoplastic tumor. Using an autochthonous murine model of UPS and data from multiple human patient cohorts, we discovered a multifaceted mechanism wherein the transcriptional co-activator YAP1 promotes collagen VI (COLVI) deposition in the UPS TME. In turn, COLVI induces CD8+ T cell dysfunction and immune evasion by remodeling fibrillar collagen and inhibiting T cell autophagic flux. Unexpectedly, collagen I (COLI) opposed COLVI in this setting, promoting CD8+ T cell function and acting as a tumor suppressor. Thus, CD8+ T cell responses in sarcoma depend upon oncogene-mediated ECM composition and remodeling.

Sarcoma Cells Secrete Hypoxia-Modified Collagen VI to Weaken the Lung Endothelial Barrier and Promote Metastasis.

Intratumoral hypoxia correlates with metastasis and poor survival in patients with sarcoma. Using an impedance sensing assay and a zebrafish intravital microinjection model, we demonstrated here that the hypoxia-inducible collagen-modifying enzyme lysyl hydroxylase PLOD2 and its substrate collagen type VI (COLVI) weaken the lung endothelial barrier and promote transendothelial migration. Mechanistically, hypoxia-induced PLOD2 in sarcoma cells modified COLVI, which was then secreted into the vasculature. Upon reaching the apical surface of lung endothelial cells, modified COLVI from tumor cells activated integrin ß1 (ITGß1). Furthermore, activated ITGß1 colocalized with Kindlin2, initiating their interaction with F-actin and prompting its polymerization. Polymerized F-actin disrupted endothelial adherens junctions and induced barrier dysfunction. Consistently, modified and secreted COLVI was required for the late stages of lung metastasis in vivo. Analysis of patient gene expression and survival data from The Cancer Genome Atlas (TCGA) revealed an association between the expression of both PLOD2 and COLVI and patient survival. Furthermore, high levels of COLVI were detected in surgically resected sarcoma metastases from patient lungs and in the blood of tumor-bearing mice. Together, these data identify a mechanism of sarcoma lung metastasis, revealing opportunities for therapeutic intervention. SIGNIFICANCE: Collagen type VI modified by hypoxia-induced PLOD2 is secreted by sarcoma cells and binds to integrin ß1 on endothelial cells to induce barrier dysfunction, which promotes sarcoma vascular dissemination and metastasis.

Comparative oncology reveals DNMT3B as a molecular vulnerability in undifferentiated pleomorphic sarcoma.

PURPOSE: Undifferentiated pleomorphic sarcoma (UPS), an aggressive subtype of soft-tissue sarcoma (STS), is exceedingly rare in humans and lacks effective, well-tolerated therapies. In contrast, STS are relatively common in canine companion animals. Thus, incorporation of veterinary patients into studies of UPS offers an exciting opportunity to develop novel therapeutic strategies for this rare human disease. Genome-wide studies have demonstrated that UPS is characterized by aberrant patterns of DNA methylation. However, the mechanisms and impact of this epigenetic modification on UPS biology and clinical behavior are poorly understood. METHODS: DNA methylation in mammalian cells is catalyzed by the canonical DNA methyltransferases DNMT1, DNMT3A and DNMT3B. Therefore, we leveraged cell lines and tissue specimens from human and canine patients, together with an orthotopic murine model, to probe the functional and clinical significance of DNMTs in UPS. RESULTS: We found that the DNA methyltransferase DNMT3B is overexpressed in UPS relative to normal mesenchymal tissues and is associated with a poor prognosis. Consistent with these findings, genetic DNMT3B depletion strongly inhibited UPS cell proliferation and tumor progression. However, existing hypomethylating agents, including the clinically approved drug 5-aza-2'-deoxycytidine (DAC) and the DNMT3B-inhibiting tool compound nanaomycin A, were ineffective in UPS due to cellular uptake and toxicity issues. CONCLUSIONS: DNMT3B represents a promising molecular susceptibility in UPS, but further development of DNMT3B-targeting strategies for these patients is required.

Virtual screening for small-molecule pathway regulators by image-profile matching.

Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

Context Matters: Response Heterogeneity to Collagen-Targeting Approaches in Desmoplastic Cancers.

The deposition of collagen-rich desmoplastic tissue is a well-documented feature of the solid tumor microenvironment (TME). However, efforts to target the desmoplastic extracellular matrix (ECM) en masse, or collagen molecules more specifically, have been met with mixed and sometimes paradoxical results. In this review, we posit that these discrepancies are due-at least in part-to the incredible diversity of the collagen superfamily. Specifically, whereas studies of "collagen-targeting" approaches frequently refer to "collagen" as a single molecule or relatively homogeneous molecular family, 28 individual collagens have been identified in mammalian tissues, each with a unique structure, supramolecular assembly pattern, tissue distribution, and/or function. Moreover, some collagen species have been shown to exert both pro- and anti-neoplastic effects in the desmoplastic TME, even within the same cancer type. Therefore, herein, we describe the diversity of the collagen family in normal tissues and highlight the context-specific roles of individual collagen molecules in desmoplastic tumors. We further discuss how this heterogeneity relates to the variable efficacy of "collagen-targeting" strategies in this setting and provide guidance for future directions in the field.

RGS12 is a novel tumor suppressor in osteosarcoma that inhibits YAP-TEAD1-Ezrin signaling.

Osteosarcoma (OS) is the most common primary malignancy of the bone that predominantly affects children and adolescents. Hippo pathway is a crucial regulator of organ size and tumorigenesis. However, how Hippo pathway regulates the occurrence of osteosarcoma is largely unknown. Here, we reported the regulator of G protein signaling protein 12 (RGS12) is a novel Hippo pathway regulator and tumor suppressor of osteosarcoma. Depletion of Rgs12 promotes osteosarcoma progression and lung metastasis in an orthotopic xenograft mouse model. Our data showed that the knockdown of RGS12 upregulates Ezrin expression through promoting the GNA12/13-RhoA-YAP pathway. Moreover, RGS12 negatively regulates the transcriptional activity of YAP/TEAD1 complex through its PDZ domain function to inhibit the expression and function of the osteosarcoma marker Ezrin. PDZ domain peptides of RGS12 can inhibit the development of intratibial tumor and lung metastases. Collectively, this study identifies that the RGS12 is a novel tumor suppressor in osteosarcoma through inhibiting YAP-TEAD1-Ezrin signaling pathway and provides a proof of principle that targeting RGS12 may be a therapeutic strategy for osteosarcoma.

Epithelial p53 Status Modifies Stromal-Epithelial Interactions During Basal-Like Breast Carcinogenesis.

Basal-like breast cancers (BBC) exhibit subtype-specific phenotypic and transcriptional responses to stroma, but little research has addressed how stromal-epithelial interactions evolve during early BBC carcinogenesis. It is also unclear how common genetic defects, such as p53 mutations, modify these stromal-epithelial interactions. To address these knowledge gaps, we leveraged the MCF10 progression series of breast cell lines (MCF10A, MCF10AT1, and MCF10DCIS) to develop a longitudinal, tissue-contextualized model of p53-deficient, pre-malignant breast. Acinus asphericity, a morphogenetic correlate of cell invasive potential, was quantified with optical coherence tomography imaging, and gene expression microarrays were performed to identify transcriptional changes associated with p53 depletion and stromal context. Co-culture with stromal fibroblasts significantly increased the asphericity of acini derived from all three p53-deficient, but not p53-sufficient, cell lines, and was associated with the upregulation of 38 genes. When considered as a multigene score, these genes were upregulated in co-culture models of invasive BBC with increasing stromal content, as well as in basal-like relative to luminal breast cancers in two large human datasets. Taken together, stromal-epithelial interactions during early BBC carcinogenesis are dependent upon epithelial p53 status, and may play important roles in the acquisition of an invasive morphologic phenotype.

Characterizing optical coherence tomography speckle fluctuation spectra of mammary organoids during suppression of intracellular motility.

BACKGROUND: An understanding of how the mammary gland responds to toxicant and drug exposures can shed light on mechanisms of breast cancer initiation/progression and therapeutic effectiveness, respectively. In this study, we employed noninvasive, label-free and high-throughput optical coherence tomography speckle fluctuation spectroscopy (OCT-SFS) to track exposure-response relationships in three-dimensional (3D) mammary epithelial organoid models. METHODS: OCT-SFS is sensitive to relatively high speed (~0.16-8 µm/min) motions of subcellular light scattering components occurring over short (~2-114 s) time scales, termed "intracellular motility." In this study, OCT speckle fluctuation spectra are quantified by two metrics: the intracellular motility amplitude, M, and frequency-dependent motility roll-off, α. OCT-SFS was performed on human mammary organoid models comprised of pre-malignant MCF10DCIS.com cells or MCF7 adenocarcinoma cells over 6 days of exposure to either a microtubule inhibitor (Paclitaxel, Taxol) or a myosin II inhibitor (Blebbistatin). Raw values of α and M were normalized to a dynamic range corresponding to fixed (0%) and live/homeostatic (100%) organoids for each cell line. RESULTS: In this work, we observed a significant decrease in both M and α of MCF10DCIS.com organoids after 24 hours of exposure to Taxol (P<0.001), and a significant decrease only in α for MCF7 organoids after 48 hours of exposure (P<0.0001). We also observed a significant decrease in both M and α of MCF7 organoids at the longest exposure time of 6 days to Blebbistatin (P<0.0001), and a significant decrease only in M for MCF10DCIS.com organoids after 24 hours of exposure (P<0.01). CONCLUSIONS: OCT-SFS revealed cell line-specific response patterns, in terms of intracellular motility, to different motility suppression mechanisms. This provides a foundation for future OCT-SFS studies of longitudinal responses of the mammary gland in toxicology and drug research.

TGFß and Hippo Pathways Cooperate to Enhance Sarcomagenesis and Metastasis through the Hyaluronan-Mediated Motility Receptor (HMMR).

High-grade sarcomas are metastatic and pose a serious threat to patient survival. Undifferentiated pleomorphic sarcoma (UPS) is a particularly dangerous and relatively common sarcoma subtype diagnosed in adults. UPS contains large quantities of extracellular matrix (ECM) including hyaluronic acid (HA), which is linked to metastatic potential. Consistent with these observations, expression of the HA receptor, hyaluronan-mediated motility receptor (HMMR/RHAMM), is tightly controlled in normal tissues and upregulated in UPS. Moreover, HMMR expression correlates with poor clinical outcome in these patients. Deregulation of the tumor-suppressive Hippo pathway is also linked to poor outcome in these patients. YAP1, the transcriptional regulator and central effector of Hippo pathway, is aberrantly stabilized in UPS and was recently shown to control RHAMM expression in breast cancer cells. Interestingly, both YAP1 and RHAMM are linked to TGFß signaling. Therefore, we investigated crosstalk between YAP1 and TGFß resulting in enhanced RHAMM-mediated cell migration and invasion. We observed that HMMR expression is under the control of both YAP1 and TGFß and can be effectively targeted with small-molecule approaches that inhibit these pathways. Furthermore, we found that RHAMM expression promotes tumor cell proliferation and migration/invasion. To test these observations in a robust and quantifiable in vivo system, we developed a zebrafish xenograft assay of metastasis, which is complimentary to our murine studies. Importantly, pharmacologic inhibition of the TGFß-YAP1-RHAMM axis prevents vascular migration of tumor cells to distant sites. IMPLICATIONS: These studies reveal key metastatic signaling mechanisms and highlight potential approaches to prevent metastatic dissemination in UPS.YAP1 and TGFß cooperatively enhance proliferation and migration/invasion of UPS and fibrosarcomas.

Vascular density of histologically benign breast tissue from women with breast cancer: associations with tissue composition and tumor characteristics.

In breast tumors, it is well established that intratumoral angiogenesis is crucial for malignant progression, but little is known about the vascular characteristics of extratumoral, cancer-adjacent breast. Genome-wide transcriptional data suggest that extratumoral microenvironments may influence breast cancer phenotypes; thus, histologic features of cancer-adjacent tissue may also have clinical implications. To this end, we developed a digital algorithm to quantitate vascular density in approximately 300 histologically benign tissue specimens from breast cancer patients enrolled in the UNC Normal Breast Study (NBS). Specimens were stained for CD31, and vascular content was compared to demographic variables, tissue composition metrics, and tumor molecular features. We observed that the vascular density of cancer-adjacent breast was significantly higher in older and obese women, and was strongly associated with breast adipose tissue content. Consistent with observations that older and heavier women experience higher frequencies of ER+ disease, higher extratumoral vessel density was also significantly associated with positive prognostic tumor features such as lower stage, negative nodal status, and smaller size (<2 cm). These results reveal biological relationships between extratumoral vascular content and body size, breast tissue composition, and tumor characteristics, and suggest biological plausibility for the relationship between weight gain (and corresponding breast tissue changes) and breast cancer progression.

Myeloid Slc2a1-Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT1.

Macrophages (MΦs) are heterogeneous and metabolically flexible, with metabolism strongly affecting immune activation. A classic response to proinflammatory activation is increased flux through glycolysis with a downregulation of oxidative metabolism, whereas alternative activation is primarily oxidative, which begs the question of whether targeting glucose metabolism is a viable approach to control MΦ activation. We created a murine model of myeloid-specific glucose transporter GLUT1 (Slc2a1) deletion. Bone marrow-derived MΦs (BMDM) from Slc2a1M-/- mice failed to uptake glucose and demonstrated reduced glycolysis and pentose phosphate pathway activity. Activated BMDMs displayed elevated metabolism of oleate and glutamine, yet maximal respiratory capacity was blunted in MΦ lacking GLUT1, demonstrating an incomplete metabolic reprogramming. Slc2a1M-/- BMDMs displayed a mixed inflammatory phenotype with reductions of the classically activated pro- and anti-inflammatory markers, yet less oxidative stress. Slc2a1M-/- BMDMs had reduced proinflammatory metabolites, whereas metabolites indicative of alternative activation-such as ornithine and polyamines-were greatly elevated in the absence of GLUT1. Adipose tissue MΦs of lean Slc2a1M-/- mice had increased alternative M2-like activation marker mannose receptor CD206, yet lack of GLUT1 was not a critical mediator in the development of obesity-associated metabolic dysregulation. However, Ldlr-/- mice lacking myeloid GLUT1 developed unstable atherosclerotic lesions. Defective phagocytic capacity in Slc2a1M-/- BMDMs may have contributed to unstable atheroma formation. Together, our findings suggest that although lack of GLUT1 blunted glycolysis and the pentose phosphate pathway, MΦ were metabolically flexible enough that inflammatory cytokine release was not dramatically regulated, yet phagocytic defects hindered MΦ function in chronic diseases.

TP53 protein levels, RNA-based pathway assessment, and race among invasive breast cancer cases.

Mutations in tumor suppressor TP53 have been inconsistently linked to breast cancer risk factors and survival. Immunohistochemistry (IHC) staining, a primary clinical means of TP53 mutation determination, only detects mutations that facilitate protein accumulation (e.g., missense mutations). RNA-based pathway methods capture functional status and may aid in understanding the role of TP53 function in racial disparities of breast cancer. TP53 status was assessed among invasive breast cancer cases from the Carolina Breast Cancer Study (CBCS) (2008-2013) using IHC and an established RNA-based TP53 signature (CBCS and The Cancer Genome Atlas (TCGA)). Frequency of TP53 status (IHC, RNA-based) was estimated in association with tumor characteristics, PAM50 intrinsic subtype, age, and race using relative frequency differences (RFDs) and 95% confidence intervals (95% CI) as the measure of association. Approximately 60% of basal-like tumors were TP53 protein positive (IHC), while nearly 100% were TP53 mutant-like (RNA). Luminal A tumors had low frequency of TP53 positivity (IHC: 7.9%) and mutant-like status (RNA: 1.7%). Mutant-like TP53 (RNA) was strongly associated with age ≤50 years, high tumor grade, advanced stage of disease, large tumor size, and basal-like and HER2 intrinsic subtypes. Black race was strongly associated with TP53 mutant-like status (RNA) (RFD: 24.8%, 95% CI: 20.5, 29.0) even after adjusting for age, grade, stage (RFD: 11.3%; 95% CI: 7.6, 15.0). Associations were attenuated and non-significant when measured by IHC. IHC-based TP53 status is an insensitive measurement of TP53 functional status. RNA-based methods suggest a role for TP53 in tumor prognostic features and racial disparities.

Quantification of the Effect of Toxicants on the Intracellular Kinetic Energy and Cross-Sectional Area of Mammary Epithelial Organoids by OCT Fluctuation Spectroscopy.

The ability to assess toxicant exposures of 3D in vitro mammary models that recapitulate the tissue microenvironment can aid in our understanding of environmental exposure risk over time. Longitudinal studies of 3D model systems, however, are cumbersome and suffer from a lack of high-throughput toxicological assays. In this study, we establish a noninvasive and label-free optical coherence tomography (OCT)-based imaging platform for tracking exposure-response relationships in 3D human mammary epithelial organoid models. The OCT-based assay includes metrics that quantify organoid intracellular kinetic energy and cross-sectional area (CSA). We compare the results to those obtained using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) mitochondrial dye conversion assay. Both estrogen receptor (ER)-positive (MCF7) and ER-negative (MCF10DCIS.com) breast cell lines were studied, beginning one hour after exposure and continuing for several days. Six days of exposure to 17ß-estradiol or the selective ER modulator 4-hydroxytamoxifen respectively increased or decreased MCF7 organoid CSA (p < .01), consistent with the role of estrogen signaling in ER-positive mammary epithelial cell proliferation. We also observed a significant decrease in the intracellular kinetic energy of MCF10DCIS.com organoids after 24 h of exposure to doxorubicin, a cytotoxic intercalating agent that causes DNA double-strand breaks (p < .01). MTT-based metabolic activity of MCF10DCIS.com organoids after 48 h of doxorubicin exposure decreased with dose in a similar manner as OCT-based energy metrics. These results demonstrate the feasibility of an OCT-based assay to quantify mammary epithelial cell toxicant response in vitro, noninvasively, longitudinally, and in the context of tissue microenvironments, providing a new high-throughput screening tool for toxicological studies.

Contribution of Adipose Tissue to Development of Cancer.

Solid tumor growth and metastasis require the interaction of tumor cells with the surrounding tissue, leading to a view of tumors as tissue-level phenomena rather than exclusively cell-intrinsic anomalies. Due to the ubiquitous nature of adipose tissue, many types of solid tumors grow in proximate or direct contact with adipocytes and adipose-associated stromal and vascular components, such as fibroblasts and other connective tissue cells, stem and progenitor cells, endothelial cells, innate and adaptive immune cells, and extracellular signaling and matrix components. Excess adiposity in obesity both increases risk of cancer development and negatively influences prognosis in several cancer types, in part due to interaction with adipose tissue cell populations. Herein, we review the cellular and noncellular constituents of the adipose "organ," and discuss the mechanisms by which these varied microenvironmental components contribute to tumor development, with special emphasis on obesity. Due to the prevalence of breast and prostate cancers in the United States, their close anatomical proximity to adipose tissue depots, and their complex epidemiologic associations with obesity, we particularly highlight research addressing the contribution of adipose tissue to the initiation and progression of these cancer types. Obesity dramatically modifies the adipose tissue microenvironment in numerous ways, including induction of fibrosis and angiogenesis, increased stem cell abundance, and expansion of proinflammatory immune cells. As many of these changes also resemble shifts observed within the tumor microenvironment, proximity to adipose tissue may present a hospitable environment to developing tumors, providing a critical link between adiposity and tumorigenesis. © 2018 American Physiological Society. Compr Physiol 8:237-282, 2018.

MiRiad Roles for MicroRNAs in Cardiac Development and Regeneration.

Cardiac development is an exquisitely regulated process that is sensitive to perturbations in transcriptional activity and gene dosage. Accordingly, congenital heart abnormalities are prevalent worldwide, and are estimated to occur in approximately 1% of live births. Recently, small non-coding RNAs, known as microRNAs, have emerged as critical components of the cardiogenic regulatory network, and have been shown to play numerous roles in the growth, differentiation, and morphogenesis of the developing heart. Moreover, the importance of miRNA function in cardiac development has facilitated the identification of prospective therapeutic targets for patients with congenital and acquired cardiac diseases. Here, we discuss findings attesting to the critical role of miRNAs in cardiogenesis and cardiac regeneration, and present evidence regarding the therapeutic potential of miRNAs for cardiovascular diseases.

Hyperbaric oxygen preconditioning protects skin from UV-A damage.

Hyperbaric oxygen therapy (HBOT) is used for a number of applications, including the treatment of diabetic foot ulcers and CO poisoning. However, we and others have shown that HBOT can mobilize cellular antioxidant defenses, suggesting that it may also be useful under circumstances in which tissue protection from oxidative damage is desired. To test the protective properties of hyperbaric oxygen (HBO) on a tissue level, we evaluated the ability of a preconditioning treatment regimen to protect cutaneous tissue from UV-A-induced oxidative damage. Three groups of hairless SKH1-E mice were exposed to UV-A 3 days per week for 22 weeks, with two of these groups receiving an HBO pretreatment either two or four times per week. UV-A exposure increased apoptosis and proliferation of the skin tissue, indicating elevated levels of epithelial damage and repair. Pretreatment with HBO significantly reduced UV-A-induced apoptosis and proliferation. A morphometric analysis of microscopic tissue folds also showed a significant increase in skin creasing following UV-A exposure, which was prevented by HBO pretreatment. Likewise, skin elasticity was found to be greatest in the group treated with HBO four times per week. The effects of HBO were also apparent systemically as reductions in caspase-3 activity and expression were observed in the liver. Our findings support a protective function of HBO pretreatment from a direct oxidative challenge of UV-A to skin tissue. Similar protection of other tissues may likewise be achievable.