Stromal COL11A1: Mechanisms of Stroma-Driven Multidrug Resistance in Breast Cancer and Biomarker Potential

BackgroundSPOCK1, a matricellular glycoprotein, has been implicated in tumor progression, metastasis, and the tumor immune microenvironment, yet its specific roles in breast cancer (BRCA) remain unclear. This study aimed to systematically explore the expression pattern, prognostic significance, mutation landscape, immune association, and spatial localization of SPOCK1 in breast cancer through integrated multi-omics analyses.MethodsTranscriptomic, genomic, and clinical data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were utilized. Bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) analyses were conducted, including functional enrichment, immune infiltration assessments, mutation profiling, and transcription factor activity analysis. Multiplex immunohistochemistry (mIHC) was performed to validate the spatial distribution of SPOCK1+ cancer-associated fibroblasts (CAFs) within the tumor microenvironment. Statistical analyses were performed using R and GraphPad Prism.ResultsSPOCK1 was broadly overexpressed in multiple cancer types and significantly associated with poor prognosis in BRCA. High SPOCK1 expression correlated with immune checkpoint activation, enhanced immune infiltration, and enriched metastasis-related pathways such as epithelial–mesenchymal transition (EMT) and TGF-β signaling. Single-cell analysis identified CAFs as the primary cell population expressing SPOCK1, with spatial mIHC confirming their close proximity to tumor cells. Furthermore, SPOCK1-high CAFs exhibited stronger intercellular communications with malignant cells via collagen, fibronectin, and IGFBP signaling pathways, alongside distinct transcription factor and metabolic profiles. In breast cancer CAF cell lines with knockdown of ANXA2 we found that the expression of both SPOCK1 and IGF1 was reduced.ConclusionSPOCK1 serves as a critical regulator of breast cancer progression, influencing tumor metastasis and reshaping the immune microenvironment via CAF-mediated mechanisms. These findings suggest that targeting SPOCK1+ CAFs could offer new therapeutic opportunities for breast cancer treatment.

Cancer-associated fibroblasts (CAFs) play a role in tumour initiation and progression, possibly by inducing epithelial-to-mesenchymal transition (EMT), a series of cellular changes that is known to underlie the process of metastasis. The aim of this study was to determine whether CAFs and surrounding normal breast fibroblasts (NBFs) are able to induce EMT markers and functional changes in breast epithelial cancer cells. Matched pairs of CAFs and NBFs were established from fresh human breast cancer specimens and characterised by assessment of CXCL12 levels, α-smooth muscle actin (α-SMA) levels and response to doxorubicin. The fibroblasts were then co-cultured with MCF7 cells. Vimentin and E-cadherin expressions were determined in co-cultured MCF7 cells by immunofluorescence and confocal microscopy as well as by western blotting and quantitative PCR. Co-cultured MCF7 cells were also assessed functionally by invasion assay. CAFs secreted higher levels of CXCL12 and expressed higher levels of α-SMA compared with NBFs. CAFs were also less sensitive to doxorubicin as evidenced by less H2AX phosphorylation and reduced apoptosis on flow cytometric analysis of Annexin V compared with NBFs. When co-cultured with MCF7 cells, there was greater vimentin and less E-cadherin expression as well as greater invasiveness in MCF7 cells co-cultured with CAFs compared with those co-cultured with NBFs. CAFs have the ability to induce a greater degree of EMT in MCF7 cell lines, indicating that CAFs contribute to a more malignant breast cancer phenotype and their role in influencing therapy resistance should therefore be considered when treating breast cancer.

Cancer-associated fibroblasts (CAFs) are pivotal drivers of tumor progression, yet the molecular mechanisms underlying their activation remain incompletely understood. Here, we identified the TET1/SMAD4/GATA6 regulatory axis as a central mechanism governing CAF transformation and function in breast cancer. Through integrative in vitro and in vivo models, we demonstrated that TET1, an epigenetic modulator, demethylates the SMAD4 promoter, enhancing SMAD4 expression. SMAD4 transcriptionally upregulates GATA6, which amplifies TGF-β signaling by directly activating the TGF-β promoter, establishing a self-reinforcing feedforward loop critical for CAF identity and stromal-tumor crosstalk. GATA6 and TET1 were significantly upregulated in breast CAFs compared to normal fibroblasts (NFs) and TGF-β-induced CAFs. Loss- or gain-of-function experiments revealed that these regulators control CAF survival, marker expression, and secretion of pro-tumorigenic factors. Knockdown of GATA6 or TET1 reduced CAF-mediated migration and invasion of breast cancer cells in vitro, while their overexpression enhanced cancer cell aggressiveness. Mechanistically, TET1-mediated epigenetic remodeling and GATA6-driven transcriptional activation converge on the TGF-β/SMAD pathway, sustaining CAF activation. In vivo, tumors derived from GATA6- or TET1-depleted CAFs exhibited reduced growth, proliferation, and CAF engraftment, underscoring their role in tumor progression. These findings position GATA6 and TET1 as promising targets to disrupt CAF-driven tumorigenesis, offering novel strategies for breast cancer treatment. By unraveling the epigenetic-transcriptional interplay within the tumor microenvironment, this study advances our understanding of stromal reprogramming and its implications for precision oncology.

Objective To clarify the effect of cancer-associated fibroblasts (CAFs ) autophagy on metastasis of triple-negative breast cancer (TNBC) cells co-cultured with CAFs. Methods (1) Purification and identification of CAFs. CAFs and normal fibroblasts (NFs) were prospectively obtained from the cancer tissue and normal breast tissue of six patients with TNBC at stage Ⅱ or Ⅲ, who were treated in Zhujiang Hospital of Southern Medical University from January 2015 to December 2016.α-smooth muscle actin (α-SMA) was labeled by fluorescent antibody and the proportion of α-SMA positive cells was calculated. The expression of α-SMA in CAFs and NFs was determined by Western blot, respectively, to identify CAFs used for this experiment. (2) Evaluation of CAFs autophagy. Autophagic biomarkers beclin 1 and p62 were measured by Western blot in CAFs group, NFs group and CAFs group pretreated with 3-methyladenine (CAFs+ 3-MA group), respectively. (3) Identifying the migration of MDA-MB-231 and BT-549 cells caused by CAFs autophagy. The migration of MDA-MB-231 or BT-549 cells was detected by the Transwell chamber under different conditions (medium/NFs/CAFs/CAFs+ 3-MA). (4) Clarifying the effect of CAFs autophagy on epithelial-mesenchymal transition (EMT) in MDA-MB-231 or BT-549 cells. The EMT-related proteins E-cadherin, N-cadherin and vimentin were detected by Western blot in MDA-MB-231 or BT-549 cells cultured in different conditions (medium, CAFs-CM or CAFs+ 3-MA-CM). The data of normal distribution were expressed by ±s and the data of skewed distribution were expressed by M(P25-P75). Nonparametric rank-sum test of two independent samples was used to compare α-SMA expression, t test of two independent samples was used to compare the percentage of α-SMA-positive cells, and single-factor analysis of variance was used to compare the autophagic protein expression, cell migration rate and EMT-related protein expression among groups. Results (1) α-SMA-positive cells were observed under a fluorescence microscope. The result showed that the percentage of α-SMA-positive cells in CAFs was significantly higher than that in NFs [(81.11±3.95)% vs(5.11±2.37)%, t=49.49, P<0.001)]. Western blot analysis indicated that the expression of α-SMA in CAFs and NFs was 0.98(0.95-0.98) and 0.48 (0.47-0.48), respectively, suggesting a significant difference (Z=2.023, P=0.043). (2) The expression of beclin 1 was 0.99±0.03, 0.73±0.04 and 0.26±0.02 in CAFs group, NFs group and CAFs+ 3-MA group, respectively, indicating a significant difference (F=546.188, P<0.001). The expression of beclin 1 in CAFs group was significantly higher than that in CAFs+ 3-MA group (P<0.001). The expression of p62 was 0.75±0.02, 0.98±0.03 and 0.97±0.01 in CAFs group, NFs group and CAFs+ 3-MA group, respectively, indicating a significant difference (F=136.353, P<0.001). The expression of p62 expression in CAFs group was significantly lower than that in CAFs+ 3-MA group(P<0.001). (3)The number of migrated MDA-MB-231 cells was 41.67±2.78, 23.33±2.18, 22.00±1.76 and 18.00±2.12 in CAFs group, NFs group, CAFs+ 3-MA group and medium group, respectively, indicating a significant difference(F=198.374, P<0.001). The number of migrated BT-549 cells was 35.22±1.97, 22.00±2.60, 25.11±2.15 and 15.22±2.00 in CAFs group, NFs group, CAFs+ 3-MA group and medium group, respectively, indicating a significant difference(F=129.424, P<0.001). The number of migrated MDA-MB-231 or BT-549 cells in CAFs group was significantly higher than that in other three groups (P<0.050). (4) When MDA-MB-231 cells were cultured in medium, CAFs-CM or CAFs+ 3-MA-CM, Western blot analysis showed that E-cadherin expression was 0.79±0.03, 0.54±0.02 and 0.87±0.04 in 3 groups, respectively, indicating a significant difference(F=139.286, P<0.001); N-cadherin expression was 0.59±0.02, 1.00±0.02 and 0.93±0.02 in 3 groups, respectively, indicating a significant difference(F=604.905, P<0.001); vimentin expression was 0.62±0.03, 1.01±0.01 and 0.89±0.09 in 3 groups, respectively, indicating a significant difference(F=43.884, P<0.001). When BT-549 cells was cultured in medium, CAFs-CM and CAFs+ 3-MA-CM, E-cadherin expression was 1.01±0.03, 0.63±0.03 and 0.98±0.03 in 3 groups, respectively, indicating a significant difference(F=210.102, P<0.001); N-cadherin expression was 0.58±0.01, 0.94±0.04 and 0.95±0.03 in 3 groups, respectively, indicating a significant difference(F=184.477, P<0.001), vimentin expression was 0.61±0.01, 0.98±0.03 and 0.75±0.02 in 3 groups, respectively, indicating a significant difference(F=217.659, P<0.001). Conclusion CAFs can promote the migration and EMT in MDA-MB-231 and BT-549 cells, which may be related to the autophagy of CAFs. Key words: Breast neoplasms; Fibroblasts; Autophagy

There has been an increase in the amount of studies focusing on the relevance of tumour microenvironment (TME) in metastasis of breast cancer. Cancer-associated fibroblasts (CAFs) is one of the most abundant cells found in the TME that play key roles in the metastasis of cancers particularly in invasive ductal carcinoma (IDC), a common type of breast cancer. In a bid to constantly find novel treatments to address limited number of therapy that are currently available, CAFs have emerged as a target for many researches. This review aims to explore the multifaceted role of CAFs in IDC progression, highlighting their involvement in tumour growth, angiogenesis, epithelial-to-mesenchymal transition (EMT), immune modulation, and drug resistance. Additional focus was also placed on potential therapeutic benefit of targeting CAFs. A comprehensive review of recent studies on the subject CAF biology, CAF interactions within the TME, and existing therapeutic approaches targeting CAFs in IDC was conducted. Findings show that CAFs are crucial to the dynamics of the TME and they influence tumour progression. CAFs facilitate tumour progression through various mechanisms, some of which are: secretion of pro-tumorigenic factors and ECM remodelling. CAFs also play a part in suppression of immune responses. CAF-targeted drugs have shown promising results in treatment for patients. Mechanism of CAF-targeting drugs is through inhibition of TGF-β signalling and reprogramming of CAFs. However, challenges of CAF heterogeneity and off-target effects will have to be addressed. Interestingly, CAF being a key mediator of the TME in cancer also present potential diagnostic and prognostic value to assesses IDC progression. Targeting CAFs offers a promising approach for IDC diagnosis and treatment, although more research is needed to refine strategies for selective CAF targeting and overcoming therapeutic challenges.

Purpose: The tumour microenvironment plays a critical role in tumour progression, with cancer-associated fibroblasts (CAFs) in particular being involved in this process (1). Treating breast cancer cells with conditioned media from CAFs has been shown to induce epithelial-mesenchymal transition (EMT) of the breast cancer cells compared to conditioned media from normal breast fibroblasts (NBFs) (2). This study aims to identify factors released by CAFs which induce EMT in breast cancer cells. Methodology: Primary cultures of six pairs of CAFs and NBFs were established from breast cancer patient samples. Serum-free conditioned media from these cells were collected for chemokine array analysis. MCF7 breast cancer cells were treated with recombinant chemokines and their corresponding receptor antagonists. Vimentin levels, as determined by immunoblot, and cell invasiveness, as measured by transwell assay, were used as indicators of EMT. Results: Chemokine array showed that CAFs secreted more CXCL1, CXCL8, CXCL12 and CCL2 compared to matched NBFs. Recombinant CXCL8, CXCL12 and CCL2, but not CXCL1, were able to induce vimentin and to increase invasiveness in MCF7 cells. Antagonists for receptors of CXCL8, CXCL12 and CCL2 counteracted the effect of EMT induction on MCF7 cells by recombinant chemokines. Conclusion: CXCL8, CXCL12 and CCL2, which are secreted at higher levels by CAFs compared to NBFs, induced EMT and increased invasiveness in MCF7 cells. These results suggest that therapeutic targeting of these chemokines or their receptors may be inhibitory to metastasis in some breast cancers.

Background: The pancreatic ductal adenocarcinoma tumor microenvironment plays an important role in promoting the epithelial to mesenchymal transition (EMT), an early event in pancreatic cancer, involved in cancer invasiveness and in tumor progression. Among the stromal components the cancer-associated fibroblasts (CAFs) are responsible for the peculiar pancreatic tumor microenvironment and are known to be linked to the induction of EMT. The EMT process requires a dynamic remodeling of the actin cytoskeleton and we have suggested that the splicing program of hMENA, an actin regulator, play a role in EMT. Two alternatively expressed isoforms, hMENA11a and hMENAΔv6, with opposite functions in invasiveness have been described in breast cancer (Di Modugno et al PNAS 2012). hMENA expression has not been detected in normal pancreatic ducts, whereas expressed in the human pancreatic ductal adenocarcinoma (PDAC) samples, but no data are available on hMENA alternative isoform expression in this neoplasia. The aim of this study is to investigate whether TGFβ1-mediated EMT in pancreatic cancer cells is affected by hMENA overexpression and splicing and how CAFs affect this process in cancer cell lines and in human tissues. Methods: hMENA isoform expression was evaluated in PDAC tissues by immunohistochemistry using isoform specific antibodies. hMENA isoforms and EMT markers expression were characterized in human PDAC cell lines, TGFβ1-treated or untreated, by qRT-PCR and WB analysis. The effects of either hMENA isoform specific knockdown or overexpression in the TGFβ1-induced EMT were also evaluated. Pancreatic CAFs were isolated from human tissues of resected PDAC patients. The effect of the conditioned medium of cultured CAFs was evaluated on hMENA expression. In parallel, the role of CAF-cancer cell interaction on the expression of the different hMENA isoforms was analysed using a co-culture system. Results: Freshly explanted CAFs expressed the “mesenchymal” hMENAΔv6, and not hMENA11a and secreted paracrine factors involved in the induction of hMENA isoforms in tumor cells. In a panel of pancreatic cancer cell lines, hMENA11a expression correlated with an epithelial phenotype, while hMENAΔv6 expression was correlated with a mesenchymal phenotype. Interestingly, the expression of the invasive hMENAΔv6 isoform is specifically up-regulated by TGFβ1 treatment. hMENA isoform expression levels influenced molecular changes induced by TGFβ1. Thus, the hMENA11a specific silencing led to E-cadherin down-regulation that is more evident in TGFβ1 treated cells. On the contrary, hMENA11a overexpression led to a reduction of vimentin expression and to E-cadherin up-regulation. Knockdown of the endogenous hMENA/hMENAΔv6 isoform expression prevented the activation of TGFβ1 signaling and up-regulation of mesenchymal markers. In addition, hMENA/hMENAΔv6 isoform depletion impaired the TGFβ1-induced invasiveness, migration and production of MMPs. IHC analysis of PDAC tissues revealed that the epithelial hMENA11a is rarely expressed in primary pancreatic tumour, while high levels of hMENA and hMENAΔv6 isoforms were found in 75% of primary tumours analysed. Conclusions: This data suggests that the lack of the epithelial hMENA11a isoform is an early event in pancreatic cancer, provides new insights into the role of hMENA splicing in TGFβ1-mediated EMT and highlights hMENA splicing program as an attractive pathway for the development of new therapies in PDAC. Citation Format: Roberta Melchionna, Pierluigi Iapicca, Francesca Di Modugno, Paola Trono, Novella Gualtieri, Maria Grazia Diodoro, Marcella Mottolese, Gian Luca Grazi, Matteo Fassan, Aldo Scarpa, Mina J. Bissell, Paola Nisticò. The hMENA Splicing Program: An important regulator of TGFβ1-driven EMT and invasiveness in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A60.

Cancer-associated fibroblasts (CAFs) play a substantial role in the tumor microenvironment, exhibiting a strong association with the advancement of various types of cancer, including breast, pancreatic, and prostate cancer. CAFs represent the most abundant mesenchymal cell population in breast cancer. Through diverse mechanisms, including the release of cytokines and exosomes, CAFs contribute to the progression of breast cancer by influencing tumor energy metabolism, promoting angiogenesis, impairing immune cell function, and remodeling the extracellular matrix. Moreover, CAFs considerably impact the response to treatment in breast cancer. Consequently, the development of interventions targeting CAFs has emerged as a promising therapeutic approach in the management of breast cancer. This article provides an analysis of the role of CAFs in breast cancer, specifically in relation to diagnosis, treatment, drug resistance, and prognosis. The paper succinctly outlines the diverse mechanisms through which CAFs contribute to the malignant behavior of breast cancer cells, including proliferation, invasion, metastasis, and drug resistance. Furthermore, the article emphasizes the potential of CAFs as valuable tools for early diagnosis, targeted therapy, treatment resistance, and prognosis assessment in breast cancer, thereby offering novel approaches for targeted therapy and overcoming treatment resistance in this disease.

Introduction and objection: Dysregulation of transforming growth factor-β (TGF-β) and insulin-like growth factor (IGF) axis has been linked to reactive stroma dynamics in the tumor microenvironment during prostate cancer progression. IGFBP3 induction is initiated by stroma remodeling and represents a potential therapeutic target for advanced prostate cancer. A lead quinazoline-based Doxazosin® derivative, DZ-50, generated in our laboratory (US Patent # 8377948), inhibits prostate tumor growth via inducing anoikis and disrupting focal adhesions. Molecular profiling revealed that the process of epithelial-mesenchymal-transition (EMT) is targeted by DZ-50. In this study, we investigated the effect of DZ-50 on EMT landscape, EMT to mesenchymal-epithelial-transition (MET) conversion, and invasive properties of prostate cancer cells. Methods: Human prostate cancer cells LNCaP, LNCaP overexpressing TGF-β type II receptor (TβRII), and cancer associated fibroblasts (CAFs) derived from human prostate cancer specimens, were used. The antitumor effect of DZ-50 against prostate cancer epithelial cells and CAFs was evaluated using cell viability assays. Effect of the drug on EMT key regulators (including IGFBP3) was determined using RT-PCR and Western blot analysis. Drug-induced phenotypic conversions of EMT were evaluated by confocal microscopy. Impact of TGF-β from the stroma microenvironment or exogenous cytokine, on prostate tumor cell migration and invasion, was assessed in co-cultures with CAFs. The functional contribution of IGFBP3 to EMT-MET interconversion in response to DZ-50 was assessed using siRNA approaches. Results: DZ-50 induced cell death in prostate cancer epithelial cells and CAFs, in a concentration-dependent manner. DZ-50 downregulated IGFBP3 mRNA and protein expression and promoted EMT-MET conversion in both LNCaP and LNCaPTβRII cells. IGFBP3 knockdown in LNCaPTβRII cells led to E-cadherin upregulation and MET induction, implicating IGFBP3 as a potential target of DZ-50 to reverse EMT to MET. Moreover exposure to TGF-β reversed DZ-50-induced MET by upregulating IGFBP3 in LNCaPTβRII cells. Co-cultures of LNCaPTβRII with CAFs promoted prostate cancer cell invasion via TGF-β and IGFBP3, an effect that was inhibited by the drug. Conclusions: Treatment of prostate cancer cells with the novel agent DZ-50 inhibits cell migration and invasion and causes reversal of EMT to MET by regulating IGFBP3. This study integrates IGFBP3 as new signaling effector driving the antitumor action of DZ-50 via targeting the EMT-MET phenotypic landscape in the prostate tumor microenvironment. Ongoing work in pre-clinical models will establish the therapeutic value of this novel compound in advanced metastatic prostate cancer. Citation Format: Zheng Cao, Shahriar Koochekpour, Stephen E. Strup, Natasha Kyprianou. Pharmacologic interconversion of EMT to MET for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2017-2122

Tumor metastasis is the main cause of breast cancer mortality. Increasing evidence demonstrates stromal cells play pivotal roles in promoting breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that promote breast cancer progression and metastasis; one of these factors is SDF-1 (CXCL12), a soluble chemokine that is promotes chemotaxis and motility, and facilitates cancer cell motility and angiogenesis. CAFs also secrete soluble factors that activate ERK 1/2 MAPK signaling in breast cancer cells, which has been shown to promote loss of estrogen receptor (ER) in luminal breast cancer cells. Hyperactivation of MAPK signaling (hMAPK) also associates with aggressive, basal-like and HER2-positive breast cancer and poor prognosis. Recently, we identified a patient-derived hMAPK-microRNA signature indicative of poor clinical outcome that contains microRNAs known to regulate breast cancer associated genes. The vast majority of ER- breast cancers display this microRNA signature, as do a subset of ER+ breast cancers with poorer clinical outcome. We also discovered that the breast cancers that exhibit this microRNA signature display high stromal and immune infiltrate scores, suggesting that breast cancer stroma provides important contributions to this microRNA signature and the poor clinical outcomes associated with it. To study the role of CAFs and CAF-secreted factors in breast cancer progression and metastasis, we established primary breast CAF lines from ‘indolent’ breast cancers (Luminal A), and from ‘aggressive’ breast cancers (ER-/HER2 amplified; triple negative). We have demonstrated that these CAFs differentially express several members of the hMAPK-microRNA signature compared to cultured primary breast cancer cells, supporting the contribution of stroma to the signature. Importantly hMAPK-microRNAs secreted from “aggressive” CAFs can be taken up by breast cancer cells, whereupon they repress their targets. Normal human mammary fibroblasts (HMFs) and ‘indolent’ CAFs do not secrete these microRNAs. We identified a novel class of circulating cells in the blood of breast cancer patients with metastases -CAFs (cCAFs). Patients without metastases did not have these cCAFs - suggesting cCAFs may be “aggressive” CAFs that facilitate breast cancer metastasis. Patients with overt metastasis and elevated counts of cCAFs have significantly higher levels of circulating SDF-1 in their plasma, as well as differential circulating microRNAs, and specifically hMAPK-microRNAs also found secreted by “aggressive” CAFs. Our results suggest there is a hierarchy of CAFs whereby “aggressive” tumors establish “aggressive” CAFs to facilitate metastasis. We also establish a clear link between circulating CAFs and CAF-secreted factors such as SDF-1 and microRNAs with breast cancer metastasis. Citation Format: Sanket H. Shah, Phil Miller, Leah Machlin, Kelsie Medina-Saenz, Ritesh Parajuli, Marc E. Lippman, Dorraya El-Ashry. Circulating CAFs and CAF-secreted factors may be indicative of breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4396.

Activation of the tumor-associated stroma to support tumor growth is a common feature observed in different cancer entities. This principle is exemplified by cancer-associated fibroblasts (CAFs), which are educated by the tumor to shape its development across all stages. CAFs can alter the extracellular matrix (ECM) and secrete a variety of different molecules. In that manner they have the capability to affect activation, survival, proliferation, and migration of other stromal cells and cancer cell themselves. Alteration of the ECM, desmoplasia, is a common feature of breast cancer, indicating a prominent role for CAFs in shaping tumor development in the mammary gland. In this review, we summarize the multiple roles CAFs play in mammary carcinoma. We discuss experimental and clinical strategies to interfere with CAFs function in breast cancer. Moreover, we highlight the issues arising from CAFs heterogeneity and the need for further research to identify CAFs subpopulation(s) that can be targeted to improve breast cancer therapy.

Simple SummaryRecent findings have revealed the contribution of cancer-associated fibroblasts (CAF) in immune escape in breast cancer. Still, how to specifically target immunosuppressive CAF remains an unmet medical question. Here, we provide a promising therapeutic strategy by highlighting the role of CD73 in immunosuppressive CAF. By studying cohorts of breast cancer patients and performing functional assays, our study uncovers how CD73 contributes to immunosuppression by acting in a specific CAF subpopulation (referred to as CAF-S1) in breast cancer. In addition, we validate that using an anti-CD73 antibody significantly reduces CAF-S1-mediated immunosuppression, thereby highlighting a new interesting therapeutic strategy for breast cancer patients.Background: Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance. Methods and Results: Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs. Conclusions: Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

Cancer-associated fibroblasts (CAFs) significantly contribute to tumor progression and the development of resistance to therapies across a range of malignancies, notably breast cancer. This study aims to elucidate the specific role and prognostic relevance of CALU across multiple cancer types. The association between CALU expression and prognosis, along with clinical characteristics in BRCA, HNSC, KIRP, LGG, and LIHC, was analyzed using data from the TCGA, GTEx, and GEO databases. Transcriptomic analysis of TCGA BRCA project data provided insights into the interaction between CALU and epithelial-mesenchymal transition (EMT) marker genes. Using TIMER and TISCH databases, the correlation between CALU expression and tumor microenvironment infiltration was assessed, alongside an evaluation of CALU expression across various cell types. Furthermore, CALU's influence on TNBC BRCA cell lines was explored, and its expression in tumor tissues was confirmed through immunohistochemical analysis of clinical samples. This study revealed a consistent upregulation of CALU across several tumor types, including BRCA, KIRP, LIHC, HNSC, and LGG, with elevated CALU expression being associated with unfavorable prognoses. CALU expression was particularly enhanced in clinical contexts linked to poor outcomes. Genomic analysis identified copy number alterations as the principal factor driving CALU overexpression. Additionally, a positive correlation between CALU expression and CAF infiltration was observed, along with its involvement in the EMT process in both CAFs and malignant cells. In vitro experiments demonstrated that CALU is highly expressed in TNBC-BRCA cell lines, and knockdown of CALU effectively reversed EMT progression and inhibited cellular migration. Immunohistochemical analysis of clinical samples corroborated the elevated expression of CALU in tumors, along with alterations in EMT markers. This comprehensive pan-cancer analysis underscores CALU's critical role in modulating the tumor microenvironment and facilitating cell migration via the EMT pathway, identifying it as a potential therapeutic target.

Our research has uncovered a novel and significant role for Fragile-X-mental-retardation-protein (FMRP) in cancer biology, extending its well-established functions beyond neuronal development. In a recent Science publication (Zeng et al., 2022), we demonstrated that elevated FMRP levels drive immune evasion in various murine and human tumors by modulating immune cell subsets, particularly through the suppression of effector T-cells. To further elucidate the mechanisms of FMRP-mediated immune evasion, we investigated its influence on the stromal component of the tumor microenvironment. Single-cell RNA sequencing of pancreatic ductal adenocarcinoma (PDAC) tumors revealed cancer-associated fibroblasts (CAFs) as the primary stromal cell subtype expressing FMRP. Given the crucial roles that CAFs play in tumor progression and therapeutic resistance, we explored whether FMRP expression contributes to the immunosuppressive characteristics of CAFs in PDAC and breast tumors. Our findings reveal that FMRP expression is markedly upregulated in CAFs from both breast and PDAC tumors, in contrast to its absence in naïve fibroblasts from healthy tissues. Knockdown of FMRP in CAFs resulted in substantial changes in their myofibroblastic and inflammatory properties, while overexpression of FMRP in naïve fibroblasts was sufficient to induce a CAF-like phenotype. Notably, FMRP overexpression led to significantly elevated inflammatory CAF (iCAF) and myofibroblastic CAF (myCAF) signatures, highlighting its role as a key pan-CAF regulator that influences both extracellular matrix (ECM) modulation and the secretory phenotype of CAFs. We utilized the full-body knockout (KO) mice model of the Fmr1 gene, which encodes FMRP, to further delineate the functional importance of FMRP expression in CAFs in stimulating an immunosuppressive program in vivo. Through bulk RNA sequencing, we developed an FMRP- associated gene signature for CAFs and applied it to human datasets, where we observed a pronounced increase in FMRP signatures in CAFs from human PDAC and breast tumors, highlighting its translational relevance. In conclusion, our research underscores the pivotal role of FMRP in cancer biology, impacting both the immune and stromal components of the tumor microenvironment. Targeting FMRP, particularly within CAFs, emerges as a promising therapeutic strategy to improve cancer treatment outcomes. Citation Format: Simge Yucel, Nadine Fournier, Mireia Blanco Gomez, Douglas Hanahan. FMRP upregulation in cancer: Implicating FMRP-expressing cancer-associated fibroblasts in immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR009.

Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells, CAFs promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and the MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival. We have established 3 primary breast CAF lines, one from a Luminal A breast cancer, one from an ER-/Her2 amplified cancer, and one from a triple negative cancer, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability. The CAFs express several hMAPK-microRNAs preferentially compared to the DTs. In addition to the paracrine interaction of stromal and tumor cells mediated by chemokines or hormones, miRNA cross talk between stromal and tumor cells can also occur. Results: To further investigate the connection between our miRNA signature and stroma, we analyzed the TCGA and METABRIC breast cancer datasets and found that the hMAPK-miRNA identifies tumors that with signficant stromal cell infiltrate. To investigate the role of specific expression of hMAPK-miRNAs in the CAFs, CM was isolated from CAFs from "aggressive" tumors, from the "indolent" tumor, and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors secrete more exosomes and more hMAPK-microRNAs into the CM than do HMFs or CAFs from the indolent tumor. Importantly, conditioned media (CM) from the "aggressive" CAFs activate MAPK and repress ER protein, mRNA and ER 3’UTR-reporter activity in ER+ MCF-7 breast cancer cells, while HMFs and "indolent" CAFs did not. Exosomes from the "aggressive" CAFs were responsible for the ER repression. To determine if the secreted miRNA differences exhibited by the CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating hMAPK-miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Further analyses of the serum for CTCs and CAFs show that serum samples from metastatic patients had a significantly higher number of CTCs with CAFs compared to serum from patients without metastases. Conclusions: Collectively these data suggest that different CAF populations have distinct abilities to influence the phenotype and behavior of associated cancer cells and that CAF secreted hMAPK-miRNAs may play important roles in breast cancer progression. They further suggest that these CAF secreted miRNAs can be found in patient serum along with circulating CAFs. Citation Format: Sanket H Shah, Phil Miller, Zheng Ao, Emilio Issa, Katherine Drews Elger, Ram Datar, Marc Lippman, Dorraya El-Ashry. Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-15.