Regulation of Collective Metastasis by Nanolumenal Signaling.

Collective metastasis is defined as the cohesive migration and metastasis of multicellular tumor cell clusters. Disrupting various cell adhesion genes markedly reduces cluster formation and colonization efficiency, yet the downstream signals transmitted by clustering remain largely unknown. Here, we use mouse and human breast cancer models to identify a collective signal generated by tumor cell clusters supporting metastatic colonization. We show that tumor cell clusters produce the growth factor epigen and concentrate it within nanolumina-intercellular compartments sealed by cell-cell junctions and lined with microvilli-like protrusions. Epigen knockdown profoundly reduces metastatic outgrowth and switches clusters from a proliferative to a collective migratory state. Tumor cell clusters from basal-like 2, but not mesenchymal-like, triple-negative breast cancer cell lines have increased epigen expression, sealed nanolumina, and impaired outgrowth upon nanolumenal junction disruption. We propose that nanolumenal signaling could offer a therapeutic target for aggressive metastatic breast cancers.

Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis.

Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.

Point-activated ESR1Y541S has a dramatic effect on the development of sexually dimorphic organs.

Mutations in the estrogen receptor α (ERα) occur in endocrine-resistant metastatic breast cancer. However, a major gap persists with the lack of genetically tractable immune competent mouse models to study disease. Hence, we developed a Cre-inducible murine model expressing a point-activated ESR1Y541S (ESR1Y537S in humans) driven by its endogenous promoter. Germline expression of mutant ESR1Y541S reveals dramatic developmental defects in the reproductive organs, mammary glands, and bones of the mice. These observations provide critical insights into the tissue-specific roles of ERα during development and highlights the potential use of our model in further developmental and cancer studies.

The many facets of Notch signaling in breast cancer: toward overcoming therapeutic resistance.

Breast cancer is the second leading cause of cancer-related death in women and is a complex disease with high intratumoral and intertumoral heterogeneity. Such heterogeneity is a major driving force behind failure of current therapies and development of resistance. Due to the limitations of conventional therapies and inevitable emergence of acquired drug resistance (chemo and endocrine) as well as radio resistance, it is essential to design novel therapeutic strategies to improve the prognosis for breast cancer patients. Deregulated Notch signaling within the breast tumor and its tumor microenvironment (TME) is linked to poor clinical outcomes in treatment of resistant breast cancer. Notch receptors and ligands are also important for normal mammary development, suggesting the potential for conserved signaling pathways between normal mammary gland development and breast cancer. In this review, we focus on mechanisms by which Notch receptors and ligands contribute to normal mammary gland development and breast tumor progression. We also discuss how complex interactions between cancer cells and the TME may reduce treatment efficacy and ultimately lead to acquired drug or radio resistance. Potential combinatorial approaches aimed at disrupting Notch- and TME-mediated resistance that may aid in achieving in an improved patient prognosis are also highlighted.

Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER- breast cancer patients.

ASB13 inhibits breast cancer metastasis through promoting SNAI2 degradation and relieving its transcriptional repression of YAP.

Transcription factor SNAI2 plays key roles during development and has also been known to promote metastasis by inducing invasive phenotype and tumor-initiating activity of cancer cells. However, the post-translational regulation of SNAI2 is less well studied. We performed a dual-luciferase-based, genome-wide E3 ligase siRNA library screen and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells promoted cell migration and decreased F-actin polymerization, while overexpression of ASB13 suppressed lung metastasis. Furthermore, ASB13 knockout decreased YAP expression, and such regulation is dependent on an increased protein level of SNAI2, which in turn represses YAP transcription. YAP suppresses tumor progression in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, cell migration in vitro, and lung metastasis in vivo. Clinical data analysis reveals that ASB13 expression is positively correlated with improved overall survival in breast cancer patients. These findings establish ASB13 as a suppressor of breast cancer metastasis by promoting degradation of SNAI2 and relieving its transcriptional repression of YAP.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Estrogen-related receptors are targetable ROS sensors.

Excessive reactive oxygen species (ROS) can cause oxidative stress and consequently cell injury contributing to a wide range of diseases. Addressing the critical gaps in our understanding of the adaptive molecular events downstream ROS provocation holds promise for the identification of druggable metabolic vulnerabilities. Here, we unveil a direct molecular link between the activity of two estrogen-related receptor (ERR) isoforms and the control of glutamine utilization and glutathione antioxidant production. ERRα down-regulation restricts glutamine entry into the TCA cycle, while ERRγ up-regulation promotes glutamine-driven glutathione production. Notably, we identify increased ERRγ expression/activation as a hallmark of oxidative stress triggered by mitochondrial disruption or chemotherapy. Enhanced tumor antioxidant capacity is an underlying feature of human breast cancer (BCa) patients that respond poorly to treatment. We demonstrate that pharmacological inhibition of ERRγ with the selective inverse agonist GSK5182 increases antitumor efficacy of the chemotherapeutic paclitaxel on poor outcome BCa tumor organoids. Our findings thus underscore the ERRs as novel redox sensors and effectors of a ROS defense program and highlight the potential therapeutic advantage of exploiting ERRγ inhibitors for the treatment of BCa and other diseases where oxidative stress plays a central role.

Afadin cooperates with Claudin-2 to promote breast cancer metastasis.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that the PDZ-binding motif of Claudin-2 is necessary for anchorage-independent growth of cancer cells and is required for liver metastasis. Several PDZ domain-containing proteins were identified that interact with the PDZ-binding motif of Claudin-2 in liver metastatic breast cancer cells, including Afadin, Arhgap21, Pdlim2, Pdlim7, Rims2, Scrib, and ZO-1. We specifically examined the role of Afadin as a potential Claudin-2-interacting partner that promotes breast cancer liver metastasis. Afadin associates with Claudin-2, an interaction that requires the PDZ-binding motif of Claudin-2. Loss of Afadin also impairs the ability of breast cancer cells to form colonies in soft agar and metastasize to the lungs or liver. Immunohistochemical analysis of Claudin-2 and/or Afadin expression in 206 metastatic breast cancer tumors revealed that high levels of both Claudin-2 and Afadin in primary tumors were associated with poor disease-specific survival, relapse-free survival, lung-specific relapse, and liver-specific relapse. Our findings indicate that signaling downstream from a Claudin-2/Afadin complex enables the efficient formation of breast cancer metastases. Moreover, combining Claudin-2 and Afadin as prognostic markers better predicts the potential of breast cancer to metastasize to soft tissues.

CDK7 in breast cancer: mechanisms of action and therapeutic potential.

Cyclin-dependent kinase 7 (CDK7) serves as a pivotal regulator in orchestrating cellular cycle dynamics and gene transcriptional activity. Elevated expression levels of CDK7 have been ubiquitously documented across a spectrum of malignancies and have been concomitantly correlated with adverse clinical outcomes. This review delineates the biological roles of CDK7 and explicates the molecular pathways through which CDK7 exacerbates the oncogenic progression of breast cancer. Furthermore, we synthesize the extant literature to provide a comprehensive overview of the advancement of CDK7-specific small-molecule inhibitors, encapsulating both preclinical and clinical findings in breast cancer contexts. The accumulated evidence substantiates the conceptualization of CDK7 as a propitious therapeutic target in breast cancer management.

HIF-1-regulated expression of calreticulin promotes breast tumorigenesis and progression through Wnt/ß-catenin pathway activation.

Calreticulin (CALR) is a multifunctional protein that participates in various cellular processes, which include calcium homeostasis, cell adhesion, protein folding, and cancer progression. However, the role of CALR in breast cancer (BC) is unclear. Here, we report that CALR is overexpressed in BC compared with normal tissue, and its expression is correlated with patient mortality and stemness indices. CALR expression was increased in mammosphere cultures, CD24-CD44+ cells, and aldehyde dehydrogenase-expressing cells, which are enriched for breast cancer stem cells (BCSCs). Additionally, CALR knockdown led to BCSC depletion, which impaired tumor initiation and metastasis and enhanced chemosensitivity in vivo. Chromatin immunoprecipitation and reporter assays revealed that hypoxia-inducible factor 1 (HIF-1) directly activated CALR transcription in hypoxic BC cells. CALR expression was correlated with Wnt/ß-catenin pathway activation, and an activator of Wnt/ß-catenin signaling abrogated the inhibitory effect of CALR knockdown on mammosphere formation. Taken together, our results demonstrate that CALR facilitates BC progression by promoting the BCSC phenotype through Wnt/ß-catenin signaling in an HIF-1-dependent manner and suggest that CALR may represent a target for BC therapy.

Targeting Axl favors an antitumorigenic microenvironment that enhances immunotherapy responses by decreasing Hif-1α levels.

Hypoxia is an important phenomenon in solid tumors that contributes to metastasis, tumor microenvironment (TME) deregulation, and resistance to therapies. The receptor tyrosine kinase AXL is an HIF target, but its roles during hypoxic stress leading to the TME deregulation are not well defined. We report here that the mammary gland-specific deletion of Axl in a HER2+ mouse model of breast cancer leads to a normalization of the blood vessels, a proinflammatory TME, and a reduction of lung metastases by dampening the hypoxic response in tumor cells. During hypoxia, interfering with AXL reduces HIF-1α levels altering the hypoxic response leading to a reduction of hypoxia-induced epithelial-to-mesenchymal transition (EMT), invasion, and production of key cytokines for macrophages behaviors. These observations suggest that inhibition of Axl generates a suitable setting to increase immunotherapy. Accordingly, combining pharmacological inhibition of Axl with anti-PD-1 in a preclinical model of HER2+ breast cancer reduces the primary tumor and metastatic burdens, suggesting a potential therapeutic approach to manage HER2+ patients whose tumors present high hypoxic features.

Upper-Limb Movement Quality before and after Surgery in Women with Breast Cancer: An Exploratory Study.

(1) Background: This study aimed to describe upper-limb (UL) movement quality parameters in women after breast cancer surgery and to explore their clinical relevance in relation to post-surgical pain and disability. (2) Methods: UL movement quality was assessed in 30 women before and 3 weeks after surgery for breast cancer. Via accelerometer data captured from a sensor located at the distal end of the forearm on the operated side, various movement quality parameters (local dynamic stability, movement predictability, movement smoothness, movement symmetry, and movement variability) were investigated while women performed a cyclic, weighted reaching task. At both test moments, the Quick Disabilities of the Arm, Shoulder, and Hand (Quick DASH) questionnaire was filled out to assess UL disability and pain severity. (3) Results: No significant differences in movement quality parameters were found between the pre-surgical and post-surgical time points. No significant correlations between post-operative UL disability or pain severity and movement quality were found. (4) Conclusions: From this study sample, no apparent clinically relevant movement quality parameters could be derived for a cyclic, weighted reaching task. This suggests that the search for an easy-to-use, quantitative analysis tool for UL qualitative functioning to be used in research and clinical practice should continue.

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.

Muscleblind-like 1 suppresses breast cancer metastatic colonization and stabilizes metastasis suppressor transcripts.

Post-transcriptional deregulation is a defining feature of metastatic cancer. While many microRNAs have been implicated as regulators of metastatic progression, less is known about the roles and mechanisms of RNA-binding proteins in this process. We identified muscleblind-like 1 (MBNL1), a gene implicated in myotonic dystrophy, as a robust suppressor of multiorgan breast cancer metastasis. MBNL1 binds the 3' untranslated regions (UTRs) of DBNL (drebrin-like protein) and TACC1 (transforming acidic coiled-coil containing protein 1)-two genes that we implicate as metastasis suppressors. By enhancing the stability of these genes' transcripts, MBNL1 suppresses cell invasiveness. Consistent with these findings, elevated MBNL1 expression in human breast tumors is associated with reduced metastatic relapse likelihood. Our findings delineate a post-transcriptional network that governs breast cancer metastasis through RNA-binding protein-mediated transcript stabilization.

A phosphorylation-deubiquitination cascade regulates the BRCA2-RAD51 axis in homologous recombination.

Homologous recombination (HR) is one of the major DNA double-strand break (DSB) repair pathways in mammalian cells. Defects in HR trigger genomic instability and result in cancer predisposition. The defining step of HR is homologous strand exchange directed by the protein RAD51, which is recruited to DSBs by BRCA2. However, the regulation of the BRCA2-RAD51 axis remains unclear. Here we report that ubiquitination of RAD51 hinders RAD51-BRCA2 interaction, while deubiquitination of RAD51 facilitates RAD51-BRCA2 binding and RAD51 recruitment and thus is critical for proper HR. Mechanistically, in response to DNA damage, the deubiquitinase UCHL3 is phosphorylated and activated by ATM. UCHL3, in turn, deubiquitinates RAD51 and promotes the binding between RAD51 and BRCA2. Overexpression of UCHL3 renders breast cancer cells resistant to radiation and chemotherapy, while depletion of UCHL3 sensitizes cells to these treatments, suggesting a determinant role of UCHL3 in cancer therapy. Overall, we identify UCHL3 as a novel regulator of DNA repair and reveal a model in which a phosphorylation-deubiquitination cascade dynamically regulates the BRCA2-RAD51 pathway.

Clinician perspectives on patient-centered conversations about weight management with patients with early breast cancer.

BACKGROUND: The aim of this study is to gather detailed insights from breast cancer (BC) clinicians on how to have patient-centered conversations about weight and weight management with women diagnosed with early BC. A high body mass index (BMI) is a risk factor for female BC, and many women diagnosed with BC experience unhealthy weight gain after their primary treatment. The oncology team has the opportunity to discuss the importance of healthy weight for BC prognosis and survival. METHODS: The sample of community-based BC clinicians included the following: three Black clinicians, three White clinicians, and two clinicians who were neither Black nor White; six females and two males; and six MDs and two physician assistants or nurse practitioners. Semistructured telephone interviews were conducted with these clinicians regarding their experience with and insights into having healthy weight conversations during routine clinic visits. RESULTS: Clinicians noted that weight-related conversations should focus less on BMI and weight loss and more on "healthy behavior." Clinicians looked for cues from their patients as to when they were ready for "healthy weight" counseling, receptive to diet/nutrition counseling and referrals, and ready to attempt behavioral change. Clinicians noted that encouraging physical activity could be especially challenging with patients accustomed to a sedentary lifestyle. CONCLUSIONS: Clinic-based conversations about healthy weight are likely to be most productive for both patients and their treating oncologists during the post-primary treatment phase when patients are most receptive to behavioral change that enhances their prognosis and survival.

Association between major discrimination and deficit accumulation in African American cancer survivors: The Detroit Research on Cancer Survivors Study.

BACKGROUND: Discrimination can adversely affect health and accelerate aging, but little is known about these relationships in cancer survivors. This study examines associations of discrimination and aging among self-identified African American survivors. METHODS: A population-based sample of 2232 survivors 20-79 years old at diagnosis were enrolled within 5 years of breast (n = 787), colorectal (n = 227), lung (n = 223), or prostate (n = 995) cancer between 2017 and 2022. Surveys were completed post-active therapy. A deficit accumulation index measured aging-related disease and function (score range, 0-1, where <0.20 is robust, 0.20 to <0.35 is pre-frail, and 0.35+ is frail; 0.06 is a large clinically meaningful difference). The discrimination scale assessed ever experiencing major discrimination and seven types of events (score, 0-7). Linear regression tested the association of discrimination and deficit accumulation, controlling for age, time from diagnosis, cancer type, stage and therapy, and sociodemographic variables. RESULTS: Survivors were an average of 62 years old (SD, 9.6), 63.2% reported ever experiencing major discrimination, with an average of 2.4 (SD, 1.7) types of discrimination events. Only 24.4% had deficit accumulation scores considered robust (mean score, 0.30 [SD, 0.13]). Among those who reported ever experiencing major discrimination, survivors with four to seven types of discrimination events (vs. 0-1) had a large, clinically meaningful increase in adjusted deficits (0.062, p < .001) and this pattern was consistent across cancer types. CONCLUSION: African American cancer survivors have high deficit accumulated index scores, and experiences of major discrimination were positively associated with these deficits. Future studies are needed to understand the intersectionality between aging, discrimination, and cancer survivorship among diverse populations.

Hsp47 promotes cancer metastasis by enhancing collagen-dependent cancer cell-platelet interaction.

Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell-platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell-platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell-platelet interaction and cancer colonization in secondary organs.

Fibroblast-tumor cell signaling limits HER2 kinase therapy response via activation of MTOR and antiapoptotic pathways.

Despite the implementation of multiple HER2-targeted therapies, patients with advanced HER2+ breast cancer ultimately develop drug resistance. Stromal fibroblasts represent an abundant cell type in the tumor microenvironment and have been linked to poor outcomes and drug resistance. Here, we show that fibroblasts counteract the cytotoxic effects of HER2 kinase-targeted therapy in a subset of HER2+ breast cancer cell lines and allow cancer cells to proliferate in the presence of the HER2 kinase inhibitor lapatinib. Fibroblasts from primary breast tumors, normal breast tissue, and lung tissue have similar protective effects on tumor cells via paracrine factors. This fibroblast-mediated reduction in drug sensitivity involves increased expression of antiapoptotic proteins and sustained activation of the PI3K/AKT/MTOR pathway, despite inhibition of the HER2 and the RAS-ERK pathways in tumor cells. HER2 therapy sensitivity is restored in the fibroblast cocultures by combination treatment with inhibitors of MTOR or the antiapoptotic proteins BCL-XL and MCL-1. Expression of activated AKT in tumor cells recapitulates the effects of fibroblasts resulting in sustained MTOR signaling and poor lapatinib response. Lapatinib sensitivity was not altered by fibroblasts in tumor cells that exhibited sustained MTOR signaling due to a strong gain-of-function PI3KCA mutation. These findings indicate that in addition to tumor cell-intrinsic mechanisms that cause constitutive PI3K/AKT/MTOR pathway activation, secreted factors from fibroblasts can maintain this pathway in the context of HER2 inhibition. Our integrated proteomic-phenotypic approach presents a strategy for the discovery of protective mechanisms in fibroblast-rich tumors and the design of rational combination therapies to restore drug sensitivity.