In embryonic urothelium and urothelium undergoing injury repair, Sox9 is expressed in basal and intermediate cells, but not in terminally differentiated superficial cells. This expression pattern suggests that the transcription factor may antagonize urothelial differentiation, a role consonant with that seen in pre-adipocytes (36
), chondrocytes (37
), pyloric sphincter epithelial cells (38
), and early differentiation of prostate bud epithelia (7
Sox9 expression is limited to the invasive front of epithelial buds in developing prostate (7
) and lung (39
) as the buds enter surrounding mesenchyme. Tissue-specific knockout in the prostate prevents bud outgrowth, likely through defective growth, migration, or both. shRNA-mediated knockdown of SOX9 expression in UroCa cells can result in a migration defect without affecting tumor cell growth. While more studies would be needed to distinguish roles in embryonic growth versus injury repair, it may be that the proliferative role of SOX9 operates mainly in primitive embryonic cells whereas the migratory role is common to both organogenesis and injury repair.
In normal undamaged bladder, an overlying urine-blood barrier formed by superficial cells (20
) is hypothesized to protect the EGFR-enriched basal cell layer (27
) from contact with urinary EGFR ligands (especially EGF). In addition to the possibility that basal urothelial cells might respond to urinary ligands, urothelium and adjacent smooth muscle can produce EGFR ligands in response to injury, including TGFα (42
), HB-EGF, and Epiregulin (43
). It has not been previously demonstrated that injury to urothelial tissue activates EGFR signaling, and the sequelae of such activation have not been previously determined. Here we provide new evidence that HB-EGF and AREG are significantly induced by injury in urothelial tissue (), supporting an autocrine mode of action in activating EGFR. Of particular interest, HB-EGF has been found to be an autocrine growth factor for human urothelial cells (31
). This finding, coupled with the induction of this ligand by urothelial tissue injury () and evidence that HB-EGF induces SOX9 in UroCa cells (Supplementary Fig. 6E & 6F
), suggest a mechanism linking migration induced by injury to that operating in cancer.
We have discovered a role for SOX9 in cancer cell migration and invasion, indicating that SOX9 might mediate EGFR-induced cancer spread. We further provide evidence for this hypothesis by showing that SOX9 has significantly higher expression in the flat/invasive pathway of UroCa compared to non-invasive tumors or benign urothelium (). The role of SOX9 in cell migration is also consistent with the notion that urothelial cells are very mobile during injury repair and need to migrate to the superficial layer and to differentiate to heal. In UroCa cells, aberrant expression of EGFR receptors and ligands that lead to constitutive induction of SOX9 also support the invasive migratory phenotype of these cells. This notion is further supported by our recent finding that EGFR ligands, HB-EGF and NRG2, were highly expressed in a highly tumorigenic basal cell compartment in urothelial carcinoma (44
) that is also enriched for EGFR (Supplementary Fig. 2A
) and SOX9 expression (Supplementary Fig. 2D
Our findings have implications for bladder injury repair and carcinogenesis. Cancer is long been thought as a chronic wound that does not heal (45
). The ultimate source of cells for repairing injured tissue is stem/progenitors cells. For tissues with a slow turnover like urothelium, stem/progenitor cells remain quiescent. However, when injury occurs, cells can rapidly migrate (a process known as epithelial restitution), proliferate, differentiate, and remodel to heal the wound.
A common trait of both cancer and repair is the activation of signaling pathways best known for their roles in embryonic growth and patterning. We (1
) hypothesize that chronic injury acts on tissue stem cells/progenitors to permanently to activate survival, proliferation, and migration, all of which are prominent features of cancer. As part of this process, we propose that urothelial cells become cell-autonomous for EGFR activation during urothelial injury, and that such activation induces SOX9 expression to support epithelial migration and wound repair. In chronic injury, unknown genetic or epigenetic mechanisms could lock this signaling circuit in the active state, contributing to malignant transformation of urothelial cells. Future studies will be needed to expand this pathway upstream to identify mediators of sustained EGFR ligand expression and downstream to discover the molecular links between SOX9 and the migration machinery.
In the bladder, epidemiological and experimental evidence have linked human and animal bladder cancers, both squamous and urothelial (reviewed in (47
)) to chronic injury through chronic parasitic infection (48
), or environmental exposure to arsenic (49
;50) or inorganic cadmium (50
). Recent evidence indicates that arsenic can also induce EGFR ligands and activate urothelial EGFR and Erk1/2 (26
). In these situations, aberrant activation of EGFR, as well as SOX9 is anticipated based on our data.
Effective treatment of any cancer will likely require combinations of targeted therapies that overcome resistance mechanisms and redundant signaling circuits. A better understanding of inducers and effectors of this newly recognized EGFR-ERK1/2-SOX9 pathway has the potential to aid in this effort.