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Squamous cell carcinomas of the head and neck (HNSCC) and esophagus (ESCC) pose a global public health issue due to high mortality rates. Unfortunately, little progress has been made in improving patient outcomes. This is partially a result of a lack of understanding the mechanisms that drive SCC progression. Recently, Activin A signaling has been implicated in a number of cancers, yet the role of this pathway in SCC remains poorly understood. We have previously discovered that the Activin A ligand acts as a tumor suppressor when epithelial Activin receptor type IB (ACVRIB) is intact; however, this effect is lost upon ACVRIB downregulation. In the present study, we investigated the function of ACVRIB in the regulation of SCC. Using CRISPR/Cas9-mediated ACVRIB-knockout and knockdown using siRNA, we found an increased capacity to proliferate, migrate, and invade upon ACRIB loss, as ACVRIB-KO cells exhibited an altered cytoskeleton and aberrant expression of E-cadherin and integrins. Based on chemical inhibitor studies, our data suggests that these effects are mediated through ACVRIB-independent signaling via downstream activation of Smad1/5/8 and MEK/ERK. Overall, we present a novel mechanism of SCC progression upon ACVRIB loss by showing that Activin A can transduce a signal in the absence of ACVRIB., Competing Interests: None.

Proteins involved in tumor cell migration can potentially serve as markers of invasive disease. Activated Leukocyte Cell Adhesion Molecule (ALCAM) promotes adhesion, while shedding of its extracellular domain is associated with migration. We hypothesized that shed ALCAM in biofluids could be predictive of progressive disease. ALCAM expression in tumor (n = 198) and shedding in biofluids (n = 120) were measured in two separate VUMC bladder cancer cystectomy cohorts by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The primary outcome measure was accuracy of predicting 3-year overall survival (OS) with shed ALCAM compared to standard clinical indicators alone, assessed by multivariable Cox regression and concordance-indices. Validation was performed by internal bootstrap, a cohort from a second institution (n = 64), and treatment of missing data with multiple-imputation. While ALCAM mRNA expression was unchanged, histological detection of ALCAM decreased with increasing stage (P = 0.004). Importantly, urine ALCAM was elevated 17.0-fold (P < 0.0001) above non-cancer controls, correlated positively with tumor stage (P = 0.018), was an independent predictor of OS after adjusting for age, tumor stage, lymph-node status, and hematuria (HR, 1.46; 95% CI, 1.03-2.06; P = 0.002), and improved prediction of OS by 3.3% (concordance-index, 78.5% vs. 75.2%). Urine ALCAM remained an independent predictor of OS after accounting for treatment with Bacillus Calmette-Guerin, carcinoma in situ, lymph-node dissection, lymphovascular invasion, urine creatinine, and adjuvant chemotherapy (HR, 1.10; 95% CI, 1.02-1.19; P = 0.011). In conclusion, shed ALCAM may be a novel prognostic biomarker in bladder cancer, although prospective validation studies are warranted. These findings demonstrate that markers reporting on cell motility can act as prognostic indicators.

Background: Esophageal squamous cell carcinoma (ESCC) is a global public health issue, as it is the eighth most common cancer worldwide. The mechanisms behind ESCC invasion and progression are still poorly understood, and warrant further investigation into these processes and their drivers. In recent years, the ligand Activin A has been implicated as a player in the progression of a number of cancers. The objective of this study was to investigate the role of Activin A signaling in ESCC., Methods: To investigate the role Activin A plays in ESCC biology, tissue microarrays containing 200 cores from 120 ESCC patients were analyzed upon immunofluorescence staining. We utilized three-dimensional organotypic reconstruct cultures of dysplastic and esophageal squamous tumor cells lines, in the context of fibroblast-secreted Activin A, to identify the effects of Activin A on cell invasion and determine protein expression and localization in epithelial and stromal compartments by immunofluorescence. To identify the functional consequences of stromal-derived Activin A on angiogenesis, we performed endothelial tube formation assays., Results: Analysis of ESCC patient samples indicated that patients with high stromal Activin A expression had low epithelial ACVRIB, the Activin type I receptor. We found that overexpression of stromal-derived Activin A inhibited invasion of esophageal dysplastic squamous cells, ECdnT, and TE-2 ESCC cells, both positive for ACVRIB. This inhibition was accompanied by a decrease in expression of the extracellular matrix (ECM) protein fibronectin and podoplanin, which is often expressed at the leading edge during invasion. Endothelial tube formation was disrupted in the presence of conditioned media from fibroblasts overexpressing Activin A. Interestingly, ACVRIB-negative TE-11 cells did not show the prior observed effects in the context of Activin A overexpression, indicating a dependence on the presence of ACVRIB., Conclusions: We describe the first observation of an inhibitory role for Activin A in ESCC progression that is dependent on the expression of ACVRIB.

The role and function of the members of the TGFβ superfamily has been a substantial area of research focus for the last several decades. During that time, it has become apparent that aberrations in TGFβ family signaling, whether through the BMP, Activin, or TGFβ arms of the pathway, can result in tumorigenesis or contribute to its progression. Downstream signaling regulates cellular growth under normal physiological conditions yet induces diverse processes during carcinogenesis, ranging from epithelial- to-mesenchymal transition to cell migration and invasion to angiogenesis. Due to these observations, the question has been raised how to utilize and target components of these signaling pathways in cancer therapy. Given that these cascades include both ligands and receptors, there are multiple levels at which to interfere. Activin receptor-like kinases (ALKs) are a group of seven type I receptors responsible for TGFβ family signal transduction and are utilized by many ligands within the superfamily. The challenge lies in specifically targeting the often-overlapping functional effects of BMP, Activin, or TGFβ signaling during cancer progression. This review focuses on the characteristic function of the individual receptors within each subfamily and their recognized roles in cancer. We next explore the clinical utility of therapeutically targeting ALKs as some have shown partial responses in Phase I clinical trials but disappointing outcomes when used in Phase II studies. Finally, we discuss the challenges and future directions of this body of work.

Squamous cell carcinoma of the head and neck (HNSCC) accounts for more than 300,000 deaths worldwide per year as a consequence of tumor cell invasion of adjacent structures or metastasis. LIM-only protein 4 (LMO4) and LIM-domain binding protein 1 (LDB1), two directly interacting transcriptional adaptors that have important roles in normal epithelial cell differentiation, have been associated with increased metastasis, decreased differentiation, and shortened survival in carcinoma of the breast. Here, we implicate two LDB1-binding proteins, single-stranded binding protein 2 (SSBP2) and 3 (SSBP3), in controlling LMO4 and LDB1 protein abundance in HNSCC and in regulating specific tumor cell functions in this disease. First, we found that the relative abundance of LMO4, LDB1, and the two SSBPs correlated very significantly in a panel of human HNSCC cell lines. Second, expression of these proteins in tumor primaries and lymph nodes involved by metastasis were concordant in 3 of 3 sets of tissue. Third, using a Matrigel invasion and organotypic reconstruct assay, CRISPR/Cas9-mediated deletion of LDB1 in the VU-SCC-1729 cell line, which is highly invasive of basement membrane and cellular monolayers, reduced tumor cell invasiveness and migration, as well as proliferation on tissue culture plastic. Finally, inactivation of the LDB1 gene in these cells decreased growth and vascularization of xenografted human tumor cells in vivo. These data show that LMO4, LDB1, and SSBP2 and/or SSBP3 regulate metastasis, proliferation, and angiogenesis in HNSCC and provide the first evidence that SSBPs control LMO4 and LDB1 protein abundance in a cancer context., Competing Interests: The authors have declared that no competing interests exist.

The extracellular matrix protein fibronectin (FN) contributes to the structural integrity of tissues as well as the adhesive and migratory functions of cells. While FN is abundantly expressed in adult tissues, the expression of several alternatively spliced FN isoforms is restricted to embryonic development, tissue remodeling and cancer. These FN isoforms, designated ED-A and ED-B, are frequently expressed by cancer cells, tumor-associated fibroblasts and newly forming blood vessels. Using a highly sensitive collagen-based indirect ELISA, we evaluated the correlation of urinary ED-A and ED-B at time of cystectomy with overall survival in patients with high-grade bladder cancer (BCa). Detectable levels of total FN as well as ED-A and ED-B were found in urine from 85, 73 and 51 % of BCa patients, respectively. The presence of urinary ED-A was a significant independent predictor of 2-year overall survival (OS) after adjusting for age, tumor stage, lymph node stage, and urinary creatinine by multivariable Logistic Regression (p = 0.029, OR = 4.26, 95 % CI 1.16-15.71) and improved accuracy by 3.6 %. Furthermore, detection of ED-A in the urine was a significant discriminator of survival specifically in BCa patients with negative lymph node status (Log-Rank, p = 0.006; HR = 5.78, 95 % CI 1.39-24.13). Lastly, multivariable Cox proportional hazards analysis revealed that urinary ED-A was an independent prognostic indicator of 5-year OS rate for patients with BCa (p = 0.04, HR = 2.20, 95 % CI 1.04-4.69). Together, these data suggest that cancer-derived, alternatively spliced FN isoforms can act as prognostic indicators and that additional studies are warranted to assess the clinical utility of ED-A in BCa.

Our previous whole genome expression analysis of endometriomas suggested dysregulation of the ten-eleven translocation genes (TET1, TET2, and TET3), involved in converting 5- methylcytosine to 5-hydroxymethylcytosine (5-hmC). The objective of this study was to validate the expression of TET genes in ectopic and eutopic endometrium and in primary cultures of human endometrial stromal fibroblasts (HESF) during in vitro decidualization and to quantify 5-hmC levels in patients with endometriosis. Blood, eutopic endometrium, and endometriotic tissues were collected at time of gynecologic surgery. HESF cultures were created from eutopic endometrium of women without (HESF-CONTROL) and with endometriosis (HESF-ENDO) and underwent in vitro decidualization. Genomic DNA from blood and tissues underwent quantification of the absolute amount of 5-hmC using ELISA. The expression of TET1, TET2, and TET3 was decreased in endometriosis compared to non-endometriosis control eutopic endometrium. Surprisingly, the global amount of 5-hmC was higher in ectopic endometrium than control eutopic endometrium, while genomic DNA from blood of women with endometriosis contained statistically significantly less 5-hmC than women without endometriosis. Expression of TET1, TET2, and TET3 was decreased in non-decidualized HESFENDO. Upon in vitro decidualization, control HESF showed decreased expression of TET3, while decidualized HESF-ENDO showed no statistically significant change in expression of TET1, TET2, or TET3. These results indicate that the TET genes are downregulated in ectopic endometrium and in HESF-ENDO, and suggest for the first time that TET genes play a role in endometriosis. High global amounts of 5-hmC in endometriotic tissues suggest unique epigenetic regulation in these tissues.

TGFβ signaling has been implicated in the metaplasia from squamous epithelia to Barrett's esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barrett's tumorigenesis is less well established. As tumorigenesis is influenced by factors in the tumor microenvironment, such as fibroblasts and the extracellular matrix, we aimed to determine if epithelial cell-derived Activin affects initiation and progression differently than Activin signaling stimulation from a mimicked stromal source. Using Barrett's esophagus cells, CPB, and the esophageal adenocarcinoma cell lines OE33 and FLO-1, we showed that Activin reduces colony formation only in CPB cells. Epithelial cell overexpression of Activin increased cell migration and invasion in Boyden chamber assays in CPB and FLO-1 cells, which exhibited mesenchymal features such as the expression of the CD44 standard form, vimentin, and MT1-MMP. When grown in organotypic reconstructs, OE33 cells expressed E-cadherin and Keratin 8. As mesenchymal characteristics have been associated with the acquisition of stem cell-like features, we analyzed the expression and localization of SOX9, showing nuclear localization of SOX9 in esophageal CPB and FLO-1 cells.In conclusion, we show a role for autocrine Activin signaling in the regulation of colony formation, cell migration and invasion in Barrett's tumorigenesis.

Background: Tumor metastasis is responsible for 90% of cancer-related deaths. Recently, a strong link between microRNA dysregulation and human cancers has been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear., Methods: We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western blotting using overexpression and shRNA knock-down approaches. Furthermore, methylation studies were used to assess epigenetic regulation of expression. Functionally, we determined the cellular consequences using migration and invasion assays, as well as proliferation assays. Immunoprecipitation and ChIP were used to identify complex formation of SOX4 and co-repressor components., Results: Here, we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3'-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues., Conclusions: Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas.

The TGFβ signaling pathway is essential to epithelial homeostasis and is often inhibited during progression of esophageal squamous cell carcinoma. Recently, an important role for TGFβ signaling has been described in the crosstalk between epithelial and stromal cells regulating squamous tumor cell invasion in mouse models of head-and-neck squamous cell carcinoma (HNSCC). Loss of TGFβ signaling, in either compartment, leads to HNSCC however, the mechanisms involved are not well understood. Using organotypic reconstruct cultures (OTC) to model the interaction between epithelial and stromal cells that occur in dysplastic lesions, we show that loss of TGFβ signaling promotes an invasive phenotype in both fibroblast and epithelial compartments. Employing immortalized esophageal keratinocytes established to reproduce common mutations of esophageal squamous cell carcinoma, we show that treatment of OTC with inhibitors of TGFβ signaling (A83-01 or SB431542) enhances invasion of epithelial cells into a fibroblast-embedded Matrigel/collagen I matrix. Invasion induced by A83-01 is independent of proliferation but relies on protease activity and expression of ADAMTS-1 and can be altered by matrix density. This invasion was associated with increased expression of pro-inflammatory cytokines, IL1 and EGFR ligands HB-EGF and TGFα. Altering EGF signaling prevented or induced epithelial cell invasion in this model. Loss of expression of the TGFβ target gene ROBO1 suggested that chemorepulsion may regulate keratinocyte invasion. Taken together, our data show increased invasion through inhibition of TGFβ signaling altered epithelial-fibroblasts interactions, repressing markers of activated fibroblasts, and altering integrin-fibronectin interactions. These results suggest that inhibition of TGFβ signaling modulates an array of pathways that combined promote multiple aspects of tumor invasion., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In recent years, a significant amount of research has examined the controversial role of activin A in cancer. Activin A, a member of the transforming growth factor β (TGFβ) superfamily, is best characterized for its function during embryogenesis in mesoderm cell fate differentiation and reproduction. During embryogenesis, TGFβ superfamily ligands, TGFβ, bone morphogenic proteins (BMPs) and activins, act as potent morphogens. Similar to TGFβs and BMPs, activin A is a protein that is highly systemically expressed during early embryogenesis; however, post-natal expression is overall reduced and remains under strict spatiotemporal regulation. Of importance, normal post-natal expression of activin A has been implicated in the migration and invasive properties of various immune cell types, as well as endometrial cells. Aberrant activin A signaling during development results in significant morphological defects and premature mortality. Interestingly, activin A has been found to have both oncogenic and tumor suppressor roles in cancer. Investigations into the role of activin A in prostate and breast cancer has demonstrated tumor suppressive effects, while in lung and head and neck squamous cell carcinoma, it has been consistently shown that activin A expression is correlated with increased proliferation, invasion and poor patient prognosis. Activin A signaling is highly context-dependent, which is demonstrated in studies of epithelial cell tumors and the microenvironment. This review discusses normal activin A signaling in comparison to TGFb and highlights how its dysregulation contributes to cancer progression and cell invasion.

Although the etiology of squamous cell carcinomas of the oral mucosa is well understood, the cellular origin and the exact molecular mechanisms leading to their formation are not. Previously, we observed the coordinated loss of E-cadherin (CDH1) and transforming growth factor beta receptor II (TGFBR2) in esophageal squamous tumors. To investigate if the coordinated loss of Cdh1 and Tgfbr2 is sufficient to induce tumorigenesis in vivo, we developed two mouse models targeting ablation of both genes constitutively or inducibly in the oral-esophageal epithelium. We show that the loss of both Cdh1 and Tgfbr2 in both models is sufficient to induce squamous cell carcinomas with animals succumbing to the invasive disease by 18 months of age. Advanced tumors have the ability to invade regional lymph nodes and to establish distant pulmonary metastasis. The mouse tumors showed molecular characteristics of human tumors such as overexpression of Cyclin D1. We addressed the question whether TGFβ signaling may target known stem cell markers and thereby influence tumorigenesis. From our mouse and human models, we conclude that TGFβ signaling regulates key aspects of stemness and quiescence in vitro and in vivo. This provides a new explanation for the importance of TGFβ in mucosal homeostasis., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Activin A (Act A) is a member of the TGFβ superfamily. Act A and TGFβ have multiple common downstream targets and have been described to merge in their intracellular signaling cascades and function. We have previously demonstrated that coordinated loss of E-cadherin and TGFβ receptor II (TβRII) results in epithelial cell invasion. When grown in three-dimensional organotypic reconstruct cultures, esophageal keratinocytes expressing dominant-negative mutants of E-cadherin and TβRII showed activated Smad2 in the absence of functional TβRII. However, we could show that increased levels of Act A secretion was able to induce Smad2 phosphorylation. Growth factor secretion can activate autocrine and paracrine signaling, which affects crosstalk between the epithelial compartment and the surrounding microenvironment. We show that treatment with the Act A antagonist Follistatin or with a neutralizing Act A antibody can increase cell invasion in organotypic cultures in a fibroblast- and MMP-dependent manner. Similarly, suppression of Act A with shRNA increases cell invasion and tumorigenesis in vivo. Therefore, we conclude that maintaining a delicate balance of Act A expression is critical for homeostasis in the esophageal microenvironment.

Context: Recent evidence implicates the orphan nuclear receptor, nuclear receptor subfamily 2, group F, member 2 (NR2F2; chicken ovalbumin upstream promoter-transcription factor II) as both a master regulator of angiogenesis and an oncogene in prostate and other human cancers., Objective: The objective of the study was to determine whether NR2F2 plays a role in ovarian cancer and dissect its potential mechanisms of action., Design, Setting, and Patients: We examined NR2F2 expression in healthy ovary and ovarian cancers using quantitative PCR and immunohistochemistry. NR2F2 expression was targeted in established ovarian cancer cell lines to assess the impact of dysregulated NR2F2 expression in the epithelial compartment of ovarian cancers., Results: Our results indicate that NR2F2 is robustly expressed in the stroma of healthy ovary with little or no expression in epithelia lining the ovarian surface, clefts, or crypts. This pattern of NR2F2 expression was markedly disrupted in ovarian cancers, in which decreased levels of stromal expression and ectopic epithelial expression were frequently observed. Ovarian cancers with the most disrupted patterns of NR2F2 were associated with significantly shorter disease-free interval by Kaplan-Meier analysis. Targeting NR2F2 expression in established ovarian cancer cell lines enhanced apoptosis and increased proliferation. In addition, we found that NR2F2 regulates the expression of NEK2, RAI14, and multiple other genes involved in the cell cycle, suggesting potential pathways by which dysregulated expression of NR2F2 impacts ovarian cancer., Conclusions: These results uncover novel roles for NR2F2 in ovarian cancer and point to a unique scenario in which a single nuclear receptor plays potentially distinct roles in the stromal and epithelial compartments of the same tissue.