Holli A. Loomans

Intertwining of Activin A and TGFβ Signaling: Dual Roles in Cancer Progression and Cancer Cell Invasion.

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 TGFβ and highlights how its dysregulation contributes to cancer progression and cell invasion.

SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells

BackgroundTumor 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.MethodsWe 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.ResultsHere, 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.ConclusionsThus, 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.

TGFβ loss activates ADAMTS-1-mediated EGF-dependent invasion in a model of esophageal cell invasion

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.

Activin A balance regulates epithelial invasiveness and tumorigenesis

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.

Activin a signaling regulates cell invasion and proliferation in esophageal adenocarcinoma

TGFβ signaling has been implicated in the metaplasia from squamous epithelia to Barretts esophagus and, ultimately, esophageal adenocarcinoma. The role of the family member Activin A in Barretts 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 Barretts 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 Barretts tumorigenesis.

Shed urinary ALCAM is an independent prognostic biomarker of three-year overall survival after cystectomy in patients with bladder cancer.

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.

Concerted loss of TGFβ-mediated proliferation control and E-cadherin disrupts epithelial homeostasis and causes oral squamous cell carcinoma

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.

LIM-Only Protein 4 (LMO4) and LIM Domain Binding Protein 1 (LDB1) Promote Growth and Metastasis of Human Head and Neck Cancer (LMO4 and LDB1 in Head and Neck Cancer).

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.

Esophageal squamous cell carcinoma invasion is inhibited by Activin A in ACVRIB-positive cells

BackgroundEsophageal 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.MethodsTo 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.ResultsAnalysis 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.ConclusionsWe describe the first observation of an inhibitory role for Activin A in ESCC progression that is dependent on the expression of ACVRIB.

Successful Establishment of Primary Type 2 Alveolar Epithelium with 3D Organotypic Co-culture

&NA; Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three‐dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix‐embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two‐dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell‐fibroblast interactions during lung injury and repair.