Hic-5 regulates extracellular matrix-associated gene expression and cytokine secretion in cancer associated fibroblasts.

The focal adhesion protein, Hic-5 plays a key role in promoting extracellular matrix deposition and remodeling by cancer associated fibroblasts within the tumor stroma to promote breast tumor cell invasion. However, whether stromal matrix gene expression is regulated by Hic-5 is still unknown. Utilizing a constitutive Hic-5 knockout, Mouse Mammary Tumor Virus-Polyoma Middle T-Antigen spontaneous breast tumor mouse model, bulk RNAseq analysis was performed on cancer associated fibroblasts isolated from Hic-5 knockout mammary tumors. Functional network analysis highlighted a key role for Hic-5 in extracellular matrix organization, with both structural matrix genes, as well as matrix remodeling genes being differentially expressed in relation to Hic-5 expression. The subcellular distribution of the MRTF-A transcription factor and expression of a subset of MRTF-A responsive genes was also impacted by Hic-5 expression. Additionally, cytokine array analysis of conditioned media from the Hic-5 and Hic-5 knockout cancer associated fibroblasts revealed that Hic-5 is important for the secretion of several key factors that are associated with matrix remodeling, angiogenesis and immune evasion. Together, these data provide further evidence of a central role for Hic-5 expression in cancer associated fibroblasts in regulating the composition and organization of the tumor stroma microenvironment to promote breast tumor progression.

Pro-inflammatory megakaryocyte gene expression in murine models of breast cancer.

Despite abundant research demonstrating that platelets can promote tumor cell metastasis, whether primary tumors affect platelet-producing megakaryocytes remains understudied. In this study, we used a spontaneous murine model of breast cancer to show that tumor burden reduced megakaryocyte number and size and disrupted polyploidization. Single-cell RNA sequencing demonstrated that megakaryocytes from tumor-bearing mice exhibit a pro-inflammatory phenotype, epitomized by increased Ctsg, Lcn2, S100a8, and S100a9 transcripts. Protein S100A8/A9 and lipocalin-2 levels were also increased in platelets, suggesting that tumor-induced alterations to megakaryocytes are passed on to their platelet progeny, which promoted in vitro tumor cell invasion and tumor cell lung colonization to a greater extent than platelets from wild-type animals. Our study is the first to demonstrate breast cancer-induced alterations in megakaryocytes, leading to qualitative changes in platelet content that may feedback to promote tumor metastasis.

Clonal barcoding with qPCR detection enables live cell functional analyses for cancer research.

Single-cell analysis methods are valuable tools; however, current approaches do not easily enable live cell retrieval. That is a particular issue when further study of cells that were eliminated during experimentation could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and live cell functional analysis. From complex cell populations, we generate single cell-derived clonal populations, infect each with a unique molecular barcode, and retain stocks of individual barcoded clones (BCs). We develop quantitative PCR-based and next-generation sequencing methods that we employ to identify and quantify BCs in vitro and in vivo. We apply SunCatcher to various breast cancer cell lines and combine respective BCs to create versions of the original cell lines. While the heterogeneous BC pools reproduce their original parental cell line proliferation and tumor progression rates, individual BCs are phenotypically and functionally diverse. Early spontaneous metastases can also be identified and quantified. SunCatcher thus provides a rapid and sensitive approach for studying live single-cell clones and clonal evolution, and performing functional analyses.

The focal adhesion scaffold protein Hic-5 regulates vimentin organization in fibroblasts.

Focal adhesion (FA)-stimulated reorganization of the F-actin cytoskeleton regulates cellular size, shape, and mechanical properties. However, FA cross-talk with the intermediate filament cytoskeleton is poorly understood. Genetic ablation of the FA-associated scaffold protein Hic-5 in mouse cancer-associated fibroblasts (CAFs) promoted a dramatic collapse of the vimentin network, which was rescued following EGFP-Hic-5 expression. Vimentin collapse correlated with a loss of detergent-soluble vimentin filament precursors and decreased vimentin S72/S82 phosphorylation. Additionally, fluorescence recovery after photobleaching analysis indicated impaired vimentin dynamics. Microtubule (MT)-associated EB1 tracking and Western blotting of MT posttranslational modifications indicated no change in MT dynamics that could explain the vimentin collapse. However, pharmacological inhibition of the RhoGTPase Cdc42 in Hic-5 knockout CAFs rescued the vimentin collapse, while pan-formin inhibition with SMIFH2 promoted vimentin collapse in Hic-5 heterozygous CAFs. Our results reveal novel regulation of vimentin organization/dynamics by the FA scaffold protein Hic-5 via modulation of RhoGTPases and downstream formin activity.

Interferon Signaling Is Diminished with Age and Is Associated with Immune Checkpoint Blockade Efficacy in Triple-Negative Breast Cancer.

Immune checkpoint blockade (ICB) therapy, which targets T cell-inhibitory receptors, has revolutionized cancer treatment. Among the breast cancer subtypes, evaluation of ICB has been of greatest interest in triple-negative breast cancer (TNBC) due to its immunogenicity, as evidenced by the presence of tumor-infiltrating lymphocytes and elevated PD-L1 expression relative to other subtypes. TNBC incidence is equally distributed across the age spectrum, affecting 10% to 15% of women in all age groups. Here we report that increased immune dysfunction with age limits ICB efficacy in aged TNBC-bearing mice. The tumor microenvironment in both aged mice and patients with TNBC shows decreased IFN signaling and antigen presentation, suggesting failed innate immune activation with age. Triggering innate immune priming with a STING agonist restored response to ICB in aged mice. Our data implicate age-related immune dysfunction as a mechanism of ICB resistance in mice and suggest potential prognostic utility of assessing IFN-related genes in patients with TNBC receiving ICB therapy. SIGNIFICANCE: These data demonstrate for the first time that age determines the T cell-inflamed phenotype in TNBC and affects response to ICB in mice. Evaluating IFN-related genes from tumor genomic data may aid identification of patients for whom combination therapy including an IFN pathway activator with ICB may be required.This article is highlighted in the In This Issue feature, p. 1143.

Paxillin-dependent regulation of apical-basal polarity in mammary gland morphogenesis.

Establishing apical-basal epithelial cell polarity is fundamental for mammary gland duct morphogenesis during mammalian development. While the focal adhesion adapter protein paxillin is a well-characterized regulator of mesenchymal cell adhesion signaling, F-actin cytoskeleton remodeling and single cell migration, its role in epithelial tissue organization and mammary gland morphogenesis in vivo has not been investigated. Here, using a newly developed paxillin conditional knockout mouse model with targeted ablation in the mammary epithelium, in combination with ex vivo three-dimensional organoid and acini cultures, we identify new roles for paxillin in the establishment of apical-basal epithelial cell polarity and lumen formation, as well as mammary gland duct diameter and branching. Paxillin is shown to be required for the integrity and apical positioning of the Golgi network, Par complex and the Rab11/MyoVb trafficking machinery. Paxillin depletion also resulted in reduced levels of apical acetylated microtubules, and rescue experiments with the HDAC6 inhibitor tubacin highlight the central role for paxillin-dependent regulation of HDAC6 activity and associated microtubule acetylation in controlling epithelial cell apical-basal polarity and tissue branching morphogenesis.

Hic-5 expression is a major indicator of cancer cell morphology, migration, and plasticity in three-dimensional matrices.

The focal adhesion proteins Hic-5 and paxillin have been previously identified as key regulators of MDA-MB-231 breast cancer cell migration and morphologic mesenchymal-amoeboid plasticity in three-dimensional (3D) extracellular matrices (ECMs). However, their respective roles in other cancer cell types have not been evaluated. Herein, utilizing 3D cell-derived matrices and fibronectin-coated one-dimensional substrates, we show that across a variety of cancer cell lines, the level of Hic-5 expression serves as the major indicator of the cells primary morphology, plasticity, and in vitro invasiveness. Domain mapping studies reveal sites critical to the functions of both Hic-5 and paxillin in regulating phenotype, while ectopic expression of Hic-5 in cell lines with low endogenous levels of the protein is sufficient to induce a Rac1-dependent mesenchymal phenotype and, in turn, increase amoeboid-mesenchymal plasticity and invasion. We show that the activity of vinculin, when coupled to the expression of Hic-5 is required for the mesenchymal morphology in the 3D ECM. Taken together, our results identify Hic-5 as a critical modulator of tumor cell phenotype that could be utilized in predicting tumor cell migratory and invasive behavior in vivo.

Hic-5 regulates fibrillar adhesion formation to control tumor extracellular matrix remodeling through interaction with tensin1.

The linearization of the stromal extracellular matrix (ECM) by cancer-associated fibroblasts (CAFs) facilitates tumor cell growth and metastasis. However, the mechanism by which the ECM is remodeled is not fully understood. Hic-5 (TGFß1i1), a focal adhesion scaffold protein, has previously been reported to be crucial for stromal ECM deposition and remodeling in vivo. Herein we show that CAFs lacking Hic-5 exhibit a significant reduction in the ability to form fibrillar adhesions, a specialized form of focal adhesion that promote fibronectin fibrillogenesis. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1. Furthermore, Src-dependent phosphorylation of Hic-5 facilitated the interaction with tensin1 to prevent ß1 integrin internalization and trafficking to the lysosome. The interaction between Hic-5 and tensin1 was mechanosensitive, promoting fibrillar adhesion formation and fibronectin fibrillogenesis in a rigidity-dependent fashion. Importantly, this Src-dependent mechanism was conserved in three-dimensional (3D) ECM environments. Immunohistochemistry of tensin1 showed enrichment in CAFs in vivo, which was abrogated upon deletion of Hic-5. Interestingly, elevated Hic-5 expression correlates with reduced distant metastasis-free survival in patients with basal-like, HER2+ and grade 3 tumors. Thus, we have identified Hic-5 as a crucial regulator of ECM remodeling in CAFs by promoting fibrillar adhesion formation through a novel interaction with tensin1.

Myosin 1e promotes breast cancer malignancy by enhancing tumor cell proliferation and stimulating tumor cell de-differentiation.

Despite advancing therapies, thousands of women die every year of breast cancer. Myosins, actin-dependent molecular motors, are likely to contribute to tumor formation and metastasis via their effects on cell adhesion and migration and may provide promising new targets for cancer therapies. Using the MMTV-PyMT murine model of breast cancer, we identified Myosin 1e (MYO1E) as a novel tumor promoter. Tumor latency in mice lacking MYO1E was significantly increased, and tumors formed in the absence of MYO1E displayed unusual papillary morphology, with well-differentiated layers of epithelial cells covering fibrovascular cores, rather than solid sheets of tumor cells typically observed in this cancer model. These tumors were reminiscent of papillary breast cancer in humans that is typically non-invasive and often cured by tumor excision. MYO1E-null tumors exhibited decreased expression of the markers of cell proliferation, which was recapitulated in primary tumor cells derived from MYO1E-null mice. In agreement with our findings, meta-analysis of patient survival data indicated that MYO1E expression level was associated with reduced recurrence-free survival in basal-like breast cancer. Overall, our data suggests that MYO1E contributes to breast tumor malignancy and regulates the differentiation and proliferation state of breast tumor cells.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro.

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.