TGFβ signaling networks in ovarian cancer progression and plasticity

Abstract

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. Late-stage diagnosis with significant tumor burden, accompanied by recurrence and chemotherapy resistance, contributes to this poor prognosis. These morbidities are known to be tied to events associated with epithelial-mesenchymal transition (EMT) in cancer. During EMT, localized tumor cells alter their polarity, cell-cell junctions, cell-matrix interactions, acquire motility and invasiveness and an exaggerated potential for metastatic spread. Key triggers for EMT include the Transforming Growth Factor-β (TGFβ) family of growth factors which are actively produced by a wide array of cell types within a specific tumor and metastatic environment. Although TGFβ can act as either a tumor suppressor or promoter in cancer, TGFβ exhibits its pro-tumorigenic functions at least in part via EMT. TGFβ regulates EMT both at the transcriptional and post-transcriptional levels as outlined here. Despite recent advances in TGFβ based therapeutics, limited progress has been seen for ovarian cancers that are in much need of new therapeutic strategies. Here, we summarize and discuss several recent insights into the underlying signaling mechanisms of the TGFβ isoforms in EMT in the unique metastatic environment of EOCs and the current therapeutic interventions that may be relevant.

Keywords: EMT; Metastasis; Ovarian cancer; TGFβ; Tumor microenvironment.

Fig. 1

TGFβ signaling pathways in EMT. Cleavage of the pro-domain latency-associated peptide (LAP) releases active TGFβ that can bind cell surface receptors. Cell surface receptors include the Type III receptor (TGFβRIII/betaglycan), Type II receptor (TGFβRII) and the Type I receptor (TGFβRI) (also see Table 1). TGFβ elicits cellular responses by forming ligand-receptor ternary complexes. Constitutively active TGFβRII transphosphorylates TGFβRI on Ser-thr residues, activating its kinase activity, which in turn phosphorylates SMAD2/3 (blue). Phosphorylated SMAD2/3 forms heterocomplexes (heterotrimeric or dimeric) with SMAD4 and accumulates in the nucleus to regulate expression of genes associated with EMT. SMAD7 (red) terminates signaling by increasing turnover of the kinase receptors. TGFβ also mediates cellular responses via alternate signaling pathways including (from L-R) TAK1 activation by TGFβRI mediated TRAF6 ubiquitination that can induce NF-κB, JNK, p38MAP kinase signaling; induction of PI3K and AKT-mTOR signaling; TGFβ also regulates the WNT/β-catenin pathway via AKT inhibition of GSK-3β, releasing β-catenin for nuclear accumulation; TGFβ induces activation of RhoA-ROCK signaling; activates MEK/ERK pathway via phosphorylation of ShcA by TGFβRI leading to Ras activation and downstream MAP Kinases; TGFβ promotes interaction between CDC42/RAC1 and PAK2. Activation of TGFβ signaling either via SMAD or alternate pathways can induce expression of several EMT-TFs such as TWIST, SNAIL, ZEB to promote EMT and lead to repression of E-Cadherin. miRNAs and lncRNAs also play a role in TGFβ mediated EMT by either inhibiting or stimulating EMT. miR-34a, -324-5p antagonizes TGFβ-SMAD induction of EMT whereas miR-155, −9, −10b, −181a activate EMT

Fig. 2

EMT events in ovarian cancer metastasis. In step 1. FTSECs in the fallopian tube develop STIC lesions with characteristic alterations in TP53 that develop into HGS cancers in the fallopian tube and the ovaries. Epithelial ovarian cancer (EOC) cells detach and shed into the peritoneal fluid for transcoelomic spread or enter the blood vessels leading to hematogenous metastasis. In Step 2, shed EOCs in the ascites retain epithelial characteristics, undergo EMT, or acquire mesenchymal characteristics, or enter a partial E/M state, forming anoikis resistant cell aggregates. Ascites flow facilitates aggregate attachment and spread throughout the peritoneal cavity leading to cell aggregate ‘adhesions’ to the peritoneal membrane that covers the visceral organs and pelvic and abdominal cavities. Such adhesions in Step 3 can undergo MET (reverse EMT) to acquire an epithelial phenotype enabling the cells to establish and grow at secondary sites including at the omentum. At the peritoneal interface, cancer cells invade PMCs facilitated by integrins and TGFβ, developing secondary tumors and metastasis. FTSEC - fallopian tube secretory epithelial cells, STIC - serous tubal intraepithelial carcinoma, HGSC - high grade serous cancer, EOC - epithelial ovarian cancer, MET - Mesenchymal to epithelial transition, PMCs - peritoneal mesothelial cells, TGFβ - Transforming growth factor-β

Fig. 3

Ovarian cancer metastatic environment. The peritoneal and ascites environment are tightly linked to each other as leakage through the peritoneal mesothelium drives malignant ascites accumulation. Malignant ascites is composed tumor cells (in E, M or partial E/M states), either alone or in aggregates composed additionally of immune cells (macrophages, T cells, B cells and neutrophils), fibroblasts, and endothelial cells. Additional non cellular components include multiple cytokines such as TGFβ, exosomes that carry TGFβ, its receptors and also noncoding RNAs, metabolites and proteoglycans that are secreted primarily by the peritoneal mesothelial cells. In the peritoneal mesothelium, TGFβ1 released from tumor cells and CAFs can stimulate TGFβ/SMAD signaling in PMCs driving MMT, that can potentiate vascular changes leading to leakage and altered angiogenesis. Cell aggregates via integrins adhere to MMCs promoting metastasis by ECM degradation and vascular changes. The omental environment supports cell aggregate attachment to the omental MMCs, and growth preferentially near “milky” spots composed of lymphocytes, macrophages, and adipocytes. ECM - Extra cellular matrix, MMT - Mesothelial mesenchymal transition, PMCs - Peritoneal mesothelial cells, MMCs - Mesothelial mesenchymal cells, CAF - cancer associated fibroblasts

Fig. 4

Therapeutic strategies targeting TGFβ signaling. Approaches both at the preclinical and clinical stage (see Table 2) are included to demonstrate points of inhibition. TGFβ signaling can be targeted using antibodies blocking TGFβ receptor-ligand interactions, TGFβ ligand neutralizing antibodies, soluble receptor ectodomain constructs to sequester ligands (ligand trap), small molecule inhibitors against TGFβRI receptor kinase activity, anti-integrin and anti-GARP, inhibition of TGFβ activation, RNA antisense oligonucleotides preventing TGFβ translation and at the transcriptional level using peptide inhibitors and CDK8 inhibitors