Stromal cartilage oligomeric matrix protein as a tumorigenic driver in ovarian cancer via Notch3 signaling and epithelial-to-mesenchymal transition.

BACKGROUND: Cartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein, is vital in preserving cartilage integrity. Further, its overexpression is associated with the aggressiveness of several types of solid cancers. This study investigated COMP's role in ovarian cancer, exploring clinicopathological links and mechanistic insights. METHODS: To study the association of COMP expression in cancer cells and stroma with clinicopathological features of ovarian tumor patients, we analyzed an epithelial ovarian tumor cohort by immunohistochemical analysis. Subsequently, to study the functional mechanisms played by COMP, an in vivo xenograft mouse model and several molecular biology techniques such as transwell migration and invasion assay, tumorsphere formation assay, proximity ligation assay, and RT-qPCR array were performed. RESULTS: Based on immunohistochemical analysis of epithelial ovarian tumor tissues, COMP expression in the stroma, but not in cancer cells, was linked to worse overall survival (OS) of ovarian cancer patients. A xenograft mouse model showed that carcinoma-associated fibroblasts (CAFs) expressing COMP stimulate the growth and metastasis of ovarian tumors through the secretion of COMP. The expression of COMP was upregulated in CAFs stimulated with TGF-ß. Functionally, secreted COMP by CAFs enhanced the migratory capacity of ovarian cancer cells. Mechanistically, COMP activated the Notch3 receptor by enhancing the Notch3-Jagged1 interaction. The dependency of the COMP effect on Notch was confirmed when the migration and tumorsphere formation of COMP-treated ovarian cancer cells were inhibited upon incubation with Notch inhibitors. Moreover, COMP treatment induced epithelial-to-mesenchymal transition and upregulation of active ß-catenin in ovarian cancer cells. CONCLUSION: This study suggests that COMP secretion by CAFs drives ovarian cancer progression through the induction of the Notch pathway and epithelial-to-mesenchymal transition.

Glutamine deficiency drives transforming growth factor-ß signaling activation that gives rise to myofibroblastic carcinoma-associated fibroblasts.

Tumor-promoting carcinoma-associated fibroblasts (CAFs), abundant in the mammary tumor microenvironment (TME), maintain transforming growth factor-ß (TGF-ß)-Smad2/3 signaling activation and the myofibroblastic state, the hallmark of activated fibroblasts. How myofibroblastic CAFs (myCAFs) arise in the TME and which epigenetic and metabolic alterations underlie activated fibroblastic phenotypes remain, however, poorly understood. We herein show global histone deacetylation in myCAFs present in tumors to be significantly associated with poorer outcomes in breast cancer patients. As the TME is subject to glutamine (Gln) deficiency, human mammary fibroblasts (HMFs) were cultured in Gln-starved medium. Global histone deacetylation and TGF-ß-Smad2/3 signaling activation are induced in these cells, largely mediated by class I histone deacetylase (HDAC) activity. Additionally, mechanistic/mammalian target of rapamycin complex 1 (mTORC1) signaling is attenuated in Gln-starved HMFs, and mTORC1 inhibition in Gln-supplemented HMFs with rapamycin treatment boosts TGF-ß-Smad2/3 signaling activation. These data indicate that mTORC1 suppression mediates TGF-ß-Smad2/3 signaling activation in Gln-starved HMFs. Global histone deacetylation, class I HDAC activation, and mTORC1 suppression are also observed in cultured human breast CAFs. Class I HDAC inhibition or mTORC1 activation by high-dose Gln supplementation significantly attenuates TGF-ß-Smad2/3 signaling and the myofibroblastic state in these cells. These data indicate class I HDAC activation and mTORC1 suppression to be required for maintenance of myCAF traits. Taken together, these findings indicate that Gln starvation triggers TGF-ß signaling activation in HMFs through class I HDAC activity and mTORC1 suppression, presumably inducing myCAF conversion.

Metastatic seeding of human colon cancer cell clusters expressing the hybrid epithelial/mesenchymal state.

Emerging evidence supports the theory that tumor cell clusters efficiently metastasize to distant organs. However, the roles of epithelial-to-mesenchymal transition (EMT) in metastasizing tumor cell clusters have not yet been fully elucidated. To investigate this issue, tumor fragments were dissected from 40 colorectal cancer (CRC) patients and implanted subcutaneously into immunodeficient mice. We observed that tumors developed from the tumor fragments obtained from 28 of the 40 CRC patients. The tumors were then dissociated into cell suspensions to be orthotopically injected into secondary mice. The tumors from 13 of the 28 patients progressed. Furthermore, metastases formed spontaneously in the liver and lungs from the tumor fragments obtained from 8 of these 13 patients. Moreover, employing a mathematical analysis, we showed that tumor cell clusters seeded these metastases significantly more often than did single tumor cells. Membrane E-cadherin- and nuclear ZEB1-positive tumor cells indicating the hybrid epithelial/mesenchymal state were also detected in primary tumors of various CRC patients, and in the corresponding patient-derived xenografts (PDXs) and circulating tumor cell clusters in the bloodstreams of mice. In contrast, ZEB1 staining was barely detectable in the patient-matched liver metastases presumably developing through mesenchymal-to-epithelial transition. Inhibition of E-cadherin or ZEB1 expression by shRNA notably prevented the PDX-derived tumor organoids from colonizing the liver, when injected intrasplenically into mice, indicating E-cadherin and ZEB1 expressions to be required for their metastatic colonization. Taken together, these findings suggest that the epithelial/mesenchymal state mediates metastatic seeding of human CRC cell clusters into distant organs.

Activated Fibroblast Program Orchestrates Tumor Initiation and Progression; Molecular Mechanisms and the Associated Therapeutic Strategies.

: Neoplastic epithelial cells coexist in carcinomas with various non-neoplastic stromal cells, together creating the tumor microenvironment. There is a growing interest in the cross-talk between tumor cells and stromal fibroblasts referred to as carcinoma-associated fibroblasts (CAFs), which are frequently present in human carcinomas. CAF populations extracted from different human carcinomas have been shown to possess the ability to influence the hallmarks of cancer. Indeed, several mechanisms underlying CAF-promoted tumorigenesis are elucidated. Activated fibroblasts in CAFs are characterized as alpha-smooth muscle actin-positive myofibroblasts and actin-negative fibroblasts, both of which are competent to support tumor growth and progression. There are, however, heterogeneous CAF populations presumably due to the diverse sources of their progenitors in the tumor-associated stroma. Thus, molecular markers allowing identification of bona fide CAF populations with tumor-promoting traits remain under investigation. CAFs and myofibroblasts in wound healing and fibrosis share biological properties and support epithelial cell growth, not only by remodeling the extracellular matrix, but also by producing numerous growth factors and inflammatory cytokines. Notably, accumulating evidence strongly suggests that anti-fibrosis agents suppress tumor development and progression. In this review, we highlight important tumor-promoting roles of CAFs based on their analogies with wound-derived myofibroblasts and discuss the potential therapeutic strategy targeting CAFs.

Combination of glycopyrronium and indacaterol inhibits carbachol-induced ERK5 signal in fibrotic processes.

BACKGROUND: Airway fibrosis is one of the pathological features of chronic obstructive pulmonary disease (COPD), and recent studies revealed that acetylcholine plays an important role in the development of airway remodeling by stimulating proliferation and collagen synthesis of lung fibroblasts. This study was designed to examine the effects of a long-acting muscarinic receptor antagonist (LAMA) glycopyrronium and a long-acting ß2 adrenergic receptor agonist (LABA) indacaterol on acetylcholine-mediated fibrotic responses in lung fibroblasts. METHODS: After carbachol (CCh) or transforming growth factor-ß1 (TGF-ß1) exposure, the response to glycopyrronium and indacaterol was determined in vitro in fibroblasts isolated from mild-to-moderate COPD lung tissue. The ability of fibroblasts to mediate the contraction of collagen gels was assessed. The expression of α-smooth muscle actin (α-SMA) and the phosphorylation of extracellular-signal-regulated kinase 5 (ERK5) were determined by immunoblot. TGF-ß1 was quantified by ELISA and acetylcholine was quantified by liquid chromatography tandem-mass spectrometry. RESULTS: CCh stimulated fibroblast-mediated collagen gel contraction and α-SMA expression and TGF-ß1 release by fibroblasts. Blockade of autocrine TGF-ß1 attenuated CCh-mediated fibrotic responses, while TGF-ß1 did not stimulate acetylcholine release. Glycopyrronium plus indacaterol significantly attenuated CCh- and TGF-ß1-mediated fibrotic responses through inhibition of ERK5 phosphorylation. Notably, the magnitudes of CCh- and TGF-ß1-stimulated gel contraction, CCh-induced TGF-ß1 release, and ERK5 phosphorylation were greater in fibroblasts isolated from COPD subjects than in those from non-smokers. CONCLUSIONS: CCh induced TGF-ß1 self-sustaining signaling loops by potentiating ERK5 signaling and promoted myofibroblast activity. This autocrine signaling mechanism may be an attractive therapeutic target to block the fibrotic response, which was modulated by the combination of glycopyrronium and indacaterol.

The roles of tumor- and metastasis-promoting carcinoma-associated fibroblasts in human carcinomas.

Carcinoma-associated fibroblasts (CAFs) constitute a substantial proportion of the non-neoplastic mesenchymal cell compartment in various human tumors. These fibroblasts are phenotypically converted from their progenitors via interactions with nearby cancer cells during the course of tumor progression. The resulting CAFs, in turn, support the growth and progression of carcinoma cells. These fibroblasts have a major influence on the hallmarks of carcinoma and promote tumor malignancy through the secretion of tumor-promoting growth factors, cytokines and exosomes, as well as through the remodeling of the extracellular matrix. Coevolution of CAFs and carcinoma cells during tumorigenesis is therefore essential for progression into fully malignant tumors. Recent studies have revealed the molecular mechanisms underlying CAF functions, especially in tumor invasion, metastasis and drug resistance and have highlighted the significant heterogeneity among these cells. In this review, we summarize the impacts of recently identified roles of tumor-promoting CAFs and discuss the therapeutic implications of targeting the heterotypic interactions of these fibroblasts with carcinoma cells. Graphical Abstract ᅟ.

The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients.

BACKGROUND: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. METHODS: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. RESULTS: Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. CONCLUSION: Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

Carcinoma-associated fibroblasts: non-neoplastic tumour-promoting mesenchymal cells.

Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non-transformed fibroblasts expressing α-smooth muscle actin (α-SMA). MFs together with non-MF cells present in the tumour-associated stroma are collectively referred to as carcinoma-associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour-promoting interactions with other tumour-constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour-promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells.

Autocrine TGF-beta and stromal cell-derived factor-1 (SDF-1) signaling drives the evolution of tumor-promoting mammary stromal myofibroblasts.

Much interest is currently focused on the emerging role of tumor-stroma interactions essential for supporting tumor progression. Carcinoma-associated fibroblasts (CAFs), frequently present in the stroma of human breast carcinomas, include a large number of myofibroblasts, a hallmark of activated fibroblasts. These fibroblasts have an ability to substantially promote tumorigenesis. However, the precise cellular origins of CAFs and the molecular mechanisms by which these cells evolve into tumor-promoting myofibroblasts remain unclear. Using a coimplantation breast tumor xenograft model, we show that resident human mammary fibroblasts progressively convert into CAF myofibroblasts during the course of tumor progression. These cells increasingly acquire two autocrine signaling loops, mediated by TGF-ß and SDF-1 cytokines, which both act in autostimulatory and cross-communicating fashions. These autocrine-signaling loops initiate and maintain the differentiation of fibroblasts into myofibroblasts and the concurrent tumor-promoting phenotype. Collectively, these findings indicate that the establishment of the self-sustaining TGF-ß and SDF-1 autocrine signaling gives rise to tumor-promoting CAF myofibroblasts during tumor progression. This autocrine-signaling mechanism may prove to be an attractive therapeutic target to block the evolution of tumor-promoting CAFs.

Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression.

Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target.

Serum Levels of N- and C-ERC/Mesothelin and Clinicopathological Factors in Mesothelioma Patients and Those without Mesothelioma.

Objectives: ERC/mesothelin is a glycosylphosphatidylinositol (GPI)-anchor protein expressed in mesothelioma. A precursor protein is cleaved by proteases and an N-terminal fragment (N-ERC) is extracellularly secreted. A remaining C-terminal fragment (C-ERC) is tethered on cellular membranes by the GPI-anchor, but C-ERC is also released after cleavage by proteases. We and other groups reported that serum N-/C-ERC levels are associated with stages of mesothelioma and suggested the possibility of their usefulness as diagnostic markers. However, the N-ERC level is also influenced by renal functions that are not directly associated with conditions of mesothelioma. It is not known whether other clinical factors influence serum N-/C-ERC values. Furthermore, their relationship to the amount of ERC/Mesothelin in mesothelioma is not yet validated. The objective of this study is to clarify the relationship of serum N-/C-ERC levels and the status of mesothelioma and several clinical factors. Materials and Methods: We analyzed relations of serum N-/C-ERC levels and ages, gender and other clinical factors in 522 patients without mesothelioma and examined their relation to the amount of ERC/Mesothelin in mesothelioma tissues in 13 mesothelioma cases. Results: Serum N-ERC levels were influenced by renal functions. On the contrary, those of C-ERC were not influenced by any clinical factors examined in this study and were significantly correlated with the amount of ERC/Mesothelin in mesothelioma. Conclusion: Although both markers are good indicators of treatment-responses in individual patients with mesothelioma, only C-ERC reflected the amount of ERC/Mesothelin in mesothelioma among multiple patients, possibly because N-ERC was influenced by renal functions.

Increased RUNX3 expression mediates tumor-promoting ability of human breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major stromal component of human breast cancers and often promote tumor proliferation, progression and malignancy. We previously established an experimental CAF (exp-CAF) cell line equipped with a potent tumor-promoting ability. It was generated through prolonged incubation of immortalized human mammary fibroblasts with human breast cancer cells in a tumor xenograft mouse model. RESULTS: Herein, we found that the exp-CAFs highly express Runt-related transcription factor 3 (RUNX3), while counterpart fibroblasts do not. In breast cancer patients, the proportion of RUNX3-positive stromal fibroblast-like cells tends to be higher in cancerous regions than in non-cancerous regions. These findings suggest an association of RUNX3 with CAF characteristics in human breast cancers. To investigate the functional role of RUNX3 in CAFs, the exp-CAFs with or without shRNA-directed knockdown of RUNX3 were implanted with breast cancer cells subcutaneously in immunodeficient mice. Comparison of the resulting xenograft tumors revealed that tumor growth was significantly attenuated when RUNX3 expression was suppressed in the fibroblasts. Consistently, Ki-67 and CD31 immunohistochemical staining of the tumor sections indicated reduction of cancer cell proliferation and microvessel formation in the tumors formed with the RUNX3-suppressed exp-CAFs. CONCLUSION: These results suggest that increased RUNX3 expression could contribute to the tumor-promoting ability of CAFs through mediating cancer cell growth and neoangiogenesis in human breast tumors.

Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression.

Tumours are highly complex tissues composed of carcinoma cells and surrounding stroma, which is constructed by various different types of mesenchymal cells and an extracellular matrix (ECM). Carcinoma-associated fibroblasts (CAFs), which consist of both fibroblasts and myofibroblasts, are frequently observed in the stroma of human carcinomas, and their presence in large numbers is often associated with the development of high-grade malignancies and poor prognoses. Moreover, in human tumour xenograft models, CAFs extracted from the tumour are more capable of promoting tumour growth through their interactions with carcinoma cells when compared to those isolated from non-cancerous stroma. Taken together, these observations strongly suggest that CAFs actively contribute to tumour progression. In this review we highlight the emerging roles of these cells in promoting tumourigenesis, and we discuss the molecular mechanisms underlying their tumour-promoting capabilities and their cellular origin.

Phenotypic heterogeneity, stability and plasticity in tumor-promoting carcinoma-associated fibroblasts.

Reciprocal interactions between cancer cells and stromal cells in the tumor microenvironment (TME) are essential for full-blown tumor development. Carcinoma-associated fibroblasts (CAFs) are a key component of the TME together with a wide variety of stromal cell types including vascular, inflammatory, and immune cells in the extracellular matrix. CAFs not only promote tumor growth, invasion, and metastasis, but also dampen the efficacy of various therapies including immune checkpoint inhibitors. CAFs are composed of distinct fibroblast populations presumably with diverse activated fibroblastic states and tumor-promoting phenotypes in a tumor, indicating intratumor heterogeneity in these fibroblasts. Given that CAFs have been implicated in both disease progression and therapeutic responses, elucidating the functional roles of each fibroblast population in CAFs and the molecular mechanisms mediating their phenotypic stability and plasticity in the TME would be crucial for understanding tumor biology. We herein discuss how distinct fibroblast populations comprising CAFs establish their cell identities, in terms of cells-of-origin, stimuli from the TME, and the phenotypes characteristic of activated states.

The Inhibitory Effects of Anti-ERC/Mesothelin Antibody 22A31 on Colorectal Adenocarcinoma Cells, within a Mouse Xenograft Model.

The expression of Renal Carcinoma (ERC)/mesothelin is enhanced in a variety of cancers. ERC/mesothelin contributes to cancer progression by modulating cell signals that regulate proliferation and apoptosis. Based on such biological insights, ERC/mesothelin has become a molecular target for the treatment of mesothelioma, pancreatic cancer, and ovarian cancer. Recent studies revealed about 50-60% of colorectal adenocarcinomas also express ERC/mesothelin. Therefore, colorectal cancer can also be a potential target of the treatment using an anti-ERC/mesothelin antibody. We previously demonstrated an anti-tumor effect of anti-ERC antibody 22A31 against mesothelioma. In this study, we investigated the effect of 22A31 on a colorectal adenocarcinoma cell line, HCT116. The cells were xenografted into BALB/c nu/nu mice. All mice were randomly allocated to either an antibody treatment group with 22A31 or isotype-matched control IgG1κ. We compared the volume of subsequent tumors, and tumors were pathologically assessed by immunohistochemistry. Tumors treated with 22A31 were significantly smaller than those treated with IgG1κ and contained significantly fewer mitotic cells with Ki67 staining. We demonstrated that 22A31 exhibited a growth inhibitory property on HCT116. Our results implied that ERC/mesothelin-targeted therapy might be a promising treatment for colorectal cancer.

Inhibition of RAC1 activity in cancer associated fibroblasts favours breast tumour development through IL-1ß upregulation.

Cancer-associated fibroblasts (CAFs) are highly abundant stromal components in the tumour microenvironment. These cells contribute to tumorigenesis and indeed, they have been proposed as a target for anti-cancer therapies. Similarly, targeting the Rho-GTPase RAC1 has also been suggested as a potential therapeutic target in cancer. Here, we show that targeting RAC1 activity, either pharmacologically or by genetic silencing, increases the pro-tumorigenic activity of CAFs by upregulating IL-1ß secretion. Moreover, inhibiting RAC1 activity shifts the CAF subtype to a more aggressive phenotype. Thus, as RAC1 suppresses the secretion of IL-1ß by CAFs, reducing RAC1 activity in combination with the depletion of this cytokine should be considered as an interesting therapeutic option for breast cancer in which tumour cells retain intact IL-1ß signalling.

Cancer-associated fibroblast migration in non-small cell lung cancers is modulated by increased integrin α11 expression.

Cancer-associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer-stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non-small-cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC-associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5ß1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer-stroma interactions may serve as a novel, promising anti-tumor strategy.

Mesenchymal Characteristics and Predictive Biomarkers on Circulating Tumor Cells for Therapeutic Strategy.

Metastasis-related events are the primary cause of cancer-related deaths, and circulating tumor cells (CTCs) have a pivotal role in metastatic relapse. CTCs include a variety of subtypes with different functional characteristics. Interestingly, the epithelial-mesenchymal transition (EMT) markers expressed in CTCs are strongly associated with poor clinical outcome and related to the acquisition of circulating tumor stem cell (CTSC) features. Recent studies have revealed the existence of CTC clusters, also called circulating tumor microemboli (CTM), which have a high metastatic potential. In this review, we present current opinions regarding the clinical significance of CTCs and CTM with a mesenchymal phenotype as clinical surrogate markers, and we summarize the therapeutic strategy according to phenotype characterization of CTCs in various types of cancers for future precision medicine.

Stromal SNAI2 Is Required for ERBB2 Breast Cancer Progression.

SNAI2 overexpression appears to be associated with poor prognosis in breast cancer, yet it remains unclear in which breast cancer subtypes this occurs. Here we show that excess SNAI2 is associated with a poor prognosis of luminal B HER2+/ERBB2+ breast cancers in which SNAI2 expression in the stroma but not the epithelium correlates with tumor proliferation. To determine how stromal SNAI2 might influence HER2+ tumor behavior, Snai2-deficient mice were crossed with a mouse line carrying the ErbB2/Neu protooncogene to generate HER2+/ERBB2+ breast cancer. Tumors generated in this model expressed SNAI2 in the stroma but not the epithelium, allowing for the role of stromal SNAI2 to be studied without interference from the epithelial compartment. The absence of SNAI2 in the stroma of HER2+/ERBB2+ tumors is associated with: (i) lower levels of cyclin D1 (CCND1) and reduced tumor epithelium proliferation; (ii) higher levels of AKT and a lower incidence of metastasis; (iii) lower levels of angiopoietin-2 (ANGPT2), and more necrosis. Together, these results indicate that the loss of SNAI2 in cancer-associated fibroblasts limits the production of some cytokines, which influences AKT/ERK tumor signaling and subsequent proliferative and metastatic capacity of ERBB2+ breast cancer cells. Accordingly, SNAI2 expression in the stroma enhanced the tumorigenicity of luminal B HER2+/ERBB2+ breast cancers. This work emphasizes the importance of stromal SNAI2 in breast cancer progression and patients' prognosis. SIGNIFICANCE: Stromal SNAI2 expression enhances the tumorigenicity of luminal B HER2+ breast cancers and can identify a subset of patients with poor prognosis, making SNAI2 a potential therapeutic target for this disease. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/23/5216/F1.large.jpg.

CD26 expression is attenuated by TGF-ß and SDF-1 autocrine signaling on stromal myofibroblasts in human breast cancers.

Human breast carcinoma-associated fibroblasts (CAFs) increasingly acquire both transforming growth factor-ß (TGF-ß) and stromal cell-derived factor-1 (SDF-1) signaling in an autocrine fashion during tumor progression. Such signaling mediates activated myofibroblastic and tumor-promoting properties in these fibroblasts. CD26/dipeptidyl peptidase-4 is a serine protease that cleaves various chemokines including SDF-1. Stromal CD26 expression is reportedly undetectable in human skin squamous cell carcinomas. However, whether stromal CD26 expression is also downregulated in human breast cancers and which stromal cells potentially lack CD26 expression remain elusive. To answer these questions, sections prepared from 239 human breast carcinomas were stained with antibodies against CD26 and α-smooth muscle actin (α-SMA), a marker for activated myofibroblasts. We found that tumor-associated stroma involving α-SMA-positive myofibroblasts stained negative or negligible for CD26 in 118 out of 193 (61.1%) tumors, whereas noncancerous stromal regions of the breast showed considerable staining for CD26. This decreased stromal CD26 staining in tumors also tends to be associated with poor outcomes for breast cancer patients. Moreover, we demonstrated that CD26 staining is attenuated on stromal myofibroblasts in human breast cancers. Consistently, CD26 expression is significantly downregulated in cultured CAF myofibroblasts extracted from human breast carcinomas as compared to control human mammary fibroblasts. Inhibition of TGF-ß or SDF-1 signaling in CAFs by shRNA clearly upregulated the CD26 expression. Taken together, these findings indicate that CD26 expression is attenuated by TGF-ß- and SDF-1-autocrine signaling on stromal myofibroblasts in human mammary carcinomas, and that decreased stromal CD26 expression has potential as a prognostic marker.