Tumour-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

Infiltration of CD8+ T cells into tumor cell clusters in triple-negative breast cancer.

Infiltration of [Formula: see text] T lymphocytes into solid tumors is associated with good prognosis in various types of cancer, including triple-negative breast cancer (TNBC). However, the mechanisms underlying different infiltration levels are largely unknown. Here, we have characterized the spatial profile of [Formula: see text] T cells around tumor cell clusters (tightly connected tumor cells) in the core and margin regions in TNBC patient samples. We found that in some patients, the [Formula: see text] T cell density first decreases when moving in from the boundary of the tumor cell clusters and then rises again when approaching the center. To explain various infiltration profiles, we modeled the dynamics of T cell density via partial differential equations. We spatially modulated the diffusion/chemotactic coefficients of T cells (to mimic physical barriers) or introduced the localized secretion of a diffusing T cell chemorepellent. Combining the spatial-profile analysis and the modeling led to support for the second idea; i.e., there exists a possible chemorepellent inside tumor cell clusters, which prevents [Formula: see text] T cells from infiltrating into tumor cell clusters. This conclusion was consistent with an investigation into the properties of collagen fibers which suggested that variations in desmoplastic elements does not limit infiltration of [Formula: see text] T lymphocytes, as we did not observe significant correlations between the level of T cell infiltration and fiber properties. Our work provides evidence that [Formula: see text] T cells can cross typical fibrotic barriers and thus their infiltration into tumor clusters is governed by other mechanisms possibly involving a local repellent.

The Human Adenovirus Type 5 E4orf4 Protein Targets Two Phosphatase Regulators of the Hippo Signaling Pathway.

UNLABELLED: When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits, and we have shown recently that binding of E4orf4 inhibits PP2A(B55) phosphatase activity in a dose-dependent fashion by preventing access of substrates (M. Z. Mui et al., PLoS Pathog 9:e1003742, 2013, http://dx.doi.org/10.1371/journal.ppat.1003742). While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets exist. In an attempt to identify additional targets, we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2A(B55) subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway, which controls the expression of cell growth/survival genes by dephosphorylating the YAP transcriptional coactivator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling is affected by E4orf4 interactions with PP2A(B55) and/or ASPP-PP1 phosphatases. Furthermore, knockdown of YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity, as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation. IMPORTANCE: The human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels; thus, knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been known as an essential E4orf4 target, genetic analyses indicated that others must exist. To identify additional E4orf4 targets, we performed, for the first time, a large-scale affinity purification/mass spectrometry analysis of E4orf4 binding partners. Several additional candidates were detected, including key regulators of the Hippo signaling pathway, which enhances cell viability in many cancers, and results of preliminary studies suggested a link between inhibition of Hippo signaling and E4orf4 toxicity.

HSP90 inhibitors induce GPNMB cell-surface expression by modulating lysosomal positioning and sensitize breast cancer cells to glembatumumab vedotin.

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.

Unraveling Triple-Negative Breast Cancer Tumor Microenvironment Heterogeneity: Towards an Optimized Treatment Approach.

BACKGROUND: Recent efforts of gene expression profiling analyses recognized at least four different triple-negative breast cancer (TNBC) molecular subtypes. However, little is known regarding their tumor microenvironment (TME) heterogeneity. METHODS: Here, we investigated TME heterogeneity within each TNBC molecular subtype, including immune infiltrate localization and composition together with expression of targetable immune pathways, using publicly available transcriptomic and genomic datasets from a large TNBC series totaling 1512 samples. Associations between molecular subtypes and specific features were assessed using logistic regression models. All statistical tests were two-sided. RESULTS: We demonstrated that each TNBC molecular subtype exhibits distinct TME profiles associated with specific immune, vascularization, stroma, and metabolism biological processes together with specific immune composition and localization. The immunomodulatory subtype was associated with the highest expression of adaptive immune-related gene signatures and a fully inflamed spatial pattern appearing to be the optimal candidate for immune check point inhibitors. In contrast, most mesenchymal stem-like and luminal androgen receptor tumors showed an immunosuppressive phenotype as witnessed by high expression levels of stromal signatures. Basal-like, luminal androgen receptor, and mesenchymal subtypes exhibited an immune cold phenotype associated with stromal and metabolism TME signatures and enriched in margin-restricted spatial pattern. Tumors with high chromosomal instability and copy number loss in the chromosome 5q and 15q regions, including genomic loss of major histocompatibility complex related genes, showed reduced cytotoxic activity as a plausible immune escape mechanism. CONCLUSIONS: Our results demonstrate that each TNBC subtype is associated with specific TME profiles, setting the ground for a rationale tailoring of immunotherapy in TNBC patients.

Predicting Relapse in Patients With Triple Negative Breast Cancer (TNBC) Using a Deep-Learning Approach.

The abundance and/or location of tumor infiltrating lymphocytes (TILs), especially CD8+ T cells, in solid tumors can serve as a prognostic indicator in various types of cancer. However, it is often difficult to select an appropriate threshold value in order to stratify patients into well-defined risk groups. It is also important to select appropriate tumor regions to quantify the abundance of TILs. On the other hand, machine-learning approaches can stratify patients in an unbiased and automatic fashion. Based on immunofluorescence (IF) images of CD8+ T lymphocytes and cancer cells, we develop a machine-learning approach which can predict the risk of relapse for patients with Triple Negative Breast Cancer (TNBC). Tumor-section images from 9 patients with poor outcome and 15 patients with good outcome were used as a training set. Tumor-section images of 29 patients in an independent cohort were used to test the predictive power of our algorithm. In the test cohort, 6 (out of 29) patients who belong to the poor-outcome group were all correctly identified by our algorithm; for the 23 (out of 29) patients who belong to the good-outcome group, 17 were correctly predicted with some evidence that improvement is possible if other measures, such as the grade of tumors, are factored in. Our approach does not involve arbitrarily defined metrics and can be applied to other types of cancer in which the abundance/location of CD8+ T lymphocytes/other types of cells is an indicator of prognosis.

Elevated V-ATPase Activity Following PTEN Loss Is Required for Enhanced Oncogenic Signaling in Breast Cancer.

PTEN loss-of-function contributes to hyperactivation of the PI3K pathway and to drug resistance in breast cancer. Unchecked PI3K pathway signaling increases activation of the mechanistic target of rapamycin complex 1 (mTORC1), which promotes tumorigenicity. Several studies have suggested that vacuolar (H+)-ATPase (V-ATPase) complex activity is regulated by PI3K signaling. In this study, we showed that loss of PTEN elevated V-ATPase activity. Enhanced V-ATPase activity was mediated by increased expression of the ATPase H+ transporting accessory protein 2 (ATP6AP2), also known as the prorenin receptor (PRR). PRR is cleaved into a secreted extracellular fragment (sPRR) and an intracellular fragment (M8.9) that remains associated with the V-ATPase complex. Reduced PTEN expression increased V-ATPase complex activity in a PRR-dependent manner. Breast cancer cell lines with reduced PTEN expression demonstrated increased PRR expression. Similarly, PRR expression became elevated upon PTEN deletion in a mouse model of breast cancer. Interestingly, concentration of sPRR was elevated in the plasma of patients with breast cancer and correlated with tumor burden in HER2-enriched cancers. Moreover, PRR was essential for proper HER2 receptor expression, localization, and signaling. PRR knockdown attenuated HER2 signaling and resulted in reduced Akt and ERK 1/2 phosphorylation, and in lower mTORC1 activity. Overall, our study demonstrates a mechanism by which PTEN loss in breast cancer can potentiate multiple signaling pathways through upregulation of the V-ATPase complex. IMPLICATIONS: Our study contributed to the understanding of the role of the V-ATPase complex in breast cancer cell tumorigenesis and provided a potential biomarker in breast cancer.

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.

Spatially distinct tumor immune microenvironments stratify triple-negative breast cancers.

Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B+CD8+ T cells (GzmB+CD8+ T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8+ T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8+ T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells.

Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis.

CrosstalkNet: A Visualization Tool for Differential Co-expression Networks and Communities.

Variations in physiological conditions can rewire molecular interactions between biological compartments, which can yield novel insights into gain or loss of interactions specific to perturbations of interest. Networks are a promising tool to elucidate intercellular interactions, yet exploration of these large-scale networks remains a challenge due to their high dimensionality. To retrieve and mine interactions, we developed CrosstalkNet, a user friendly, web-based network visualization tool that provides a statistical framework to infer condition-specific interactions coupled with a community detection algorithm for bipartite graphs to identify significantly dense subnetworks. As a case study, we used CrosstalkNet to mine a set of 54 and 22 gene-expression profiles from breast tumor and normal samples, respectively, with epithelial and stromal compartments extracted via laser microdissection. We show how CrosstalkNet can be used to explore large-scale co-expression networks and to obtain insights into the biological processes that govern cross-talk between different tumor compartments.Significance: This web application enables researchers to mine complex networks and to decipher novel biological processes in tumor epithelial-stroma cross-talk as well as in other studies of intercompartmental interactions. Cancer Res; 78(8); 2140-3. ©2018 AACR.

Inhibition of the Stromal p38MAPK/MK2 Pathway Limits Breast Cancer Metastases and Chemotherapy-Induced Bone Loss.

The role of the stromal compartment in tumor progression is best illustrated in breast cancer bone metastases, where the stromal compartment supports tumor growth, albeit through poorly defined mechanisms. p38MAPKα is frequently expressed in tumor cells and surrounding stromal cells, and its expression levels correlate with poor prognosis. This observation led us to investigate whether inhibition of p38MAPKα could reduce breast cancer metastases in a clinically relevant model. Orally administered, small-molecule inhibitors of p38MAPKα or its downstream kinase MK2 each limited outgrowth of metastatic breast cancer cells in the bone and visceral organs. This effect was primarily mediated by inhibition of the p38MAPKα pathway within the stromal compartment. Beyond effectively limiting metastatic tumor growth, these inhibitors reduced tumor-associated and chemotherapy-induced bone loss, which is a devastating comorbidity that drastically affects quality of life for patients with cancer. These data underscore the vital role played by stromal-derived factors in tumor progression and identify the p38MAPK-MK2 pathway as a promising therapeutic target for metastatic disease and prevention of tumor-induced bone loss.Significance: Pharmacologically targeting the stromal p38MAPK-MK2 pathway limits metastatic breast cancer growth, preserves bone quality, and extends survival. Cancer Res; 78(19); 5618-30. ©2018 AACR.

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBCs) display a complex spectrum of mutations and chromosomal aberrations. Chromosome 5q (5q) loss is detected in up to 70% of TNBCs, but little is known regarding the genetic drivers associated with this event. Here, we show somatic deletion of a region syntenic with human 5q33.2-35.3 in a mouse model of TNBC. Mechanistically, we identify KIBRA as a major factor contributing to the effects of 5q loss on tumor growth and metastatic progression. Re-expression of KIBRA impairs metastasis in vivo and inhibits tumorsphere formation by TNBC cells in vitro. KIBRA functions co-operatively with the protein tyrosine phosphatase PTPN14 to trigger mechanotransduction-regulated signals that inhibit the nuclear localization of oncogenic transcriptional co-activators YAP/TAZ. Our results argue that the selective advantage produced by 5q loss involves reduced dosage of KIBRA, promoting oncogenic functioning of YAP/TAZ in TNBC.

Identification of Interacting Stromal Axes in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a molecularly heterogeneous cancer that is difficult to treat. Despite the role it may play in tumor progression and response to therapy, microenvironmental (stromal) heterogeneity in TNBC has not been well characterized. To address this challenge, we investigated the transcriptome of tumor-associated stroma isolated from TNBC (n = 57). We identified four stromal axes enriched for T cells (T), B cells (B), epithelial markers (E), or desmoplasia (D). Our analysis method (STROMA4) assigns a score along each stromal axis for each patient and then combined the axis scores to subtype patients. Analysis of these subtypes revealed that prognostic capacity of the B, T, and E scores was governed by the D score. When compared with a previously published TNBC subtyping scheme, the STROMA4 method better captured tumor heterogeneity and predicted patient benefit from therapy with increased sensitivity. This approach produces a simple ontology that captures TNBC heterogeneity and informs how tumor-associated properties interact to affect prognosis. Cancer Res; 77(17); 4673-83. ©2017 AACR.

Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients.

Anti-cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD-/Nrf2-antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly-ubiquitination and promotes its degradation by the proteasome. ROS-mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple-Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2-antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS-mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients.

MAP3K8/TPL-2/COT is a potential predictive marker for MEK inhibitor treatment in high-grade serous ovarian carcinomas.

Ovarian cancer is a silent disease with a poor prognosis that urgently requires new therapeutic strategies. In low-grade ovarian tumours, mutations in the MAP3K BRAF gene constitutively activate the downstream kinase MEK. Here we demonstrate that an additional MAP3K, MAP3K8 (TPL-2/COT), accumulates in high-grade serous ovarian carcinomas (HGSCs) and is a potential prognostic marker for these tumours. By combining analyses on HGSC patient cohorts, ovarian cancer cells and patient-derived xenografts, we demonstrate that MAP3K8 controls cancer cell proliferation and migration by regulating key players in G1/S transition and adhesion dynamics. In addition, we show that the MEK pathway is the main pathway involved in mediating MAP3K8 function, and that MAP3K8 exhibits a reliable predictive value for the effectiveness of MEK inhibitor treatment. Our data highlight key roles for MAP3K8 in HGSC and indicate that MEK inhibitors could be a useful treatment strategy, in combination with conventional chemotherapy, for this disease.

miR-141 and miR-200a act on ovarian tumorigenesis by controlling oxidative stress response.

Although there is evidence that redox regulation has an essential role in malignancies, its impact on tumor prognosis remains unclear. Here we show crosstalk between oxidative stress and the miR-200 family of microRNAs that affects tumorigenesis and chemosensitivity. miR-141 and miR-200a target p38α and modulate the oxidative stress response. Enhanced expression of these microRNAs mimics p38α deficiency and increases tumor growth in mouse models, but it also improves the response to chemotherapeutic agents. High-grade human ovarian adenocarcinomas that accumulate miR-200a have low concentrations of p38α and an associated oxidative stress signature. The miR200a-dependent stress signature correlates with improved survival of patients in response to treatment. Therefore, the role of miR-200a in stress could be a predictive marker for clinical outcome in ovarian cancer. In addition, although oxidative stress promotes tumor growth, it also sensitizes tumors to treatment, which could account for the limited success of antioxidants in clinical trials.