Carcinoma Cells Reprogram a Wound-healing Switch to Promote Metastasis

ABSTRACT

Wound healing is a dynamic event where barrier disruption is transient and miR-198/FSTL1 molecular switch orchestrate wound re-epithelialization. However, epithelial carcinomas maintain a prolonged wound-healing phase to promote malignant transformation. Delineating the molecular mechanism we demonstrate, how epidermal growth factor (EGF) hijacks the wound-healing switch to promote metastasis of carcinoma.

Head and neck squamous cell carcinoma (HNSCC) are epithelial neoplasms arising from the stratified squamous epithelium of upper aero-digestive track. This is the sixth most common cancer worldwide and more than half a million cases are reported every year. Delayed diagnosis coupled with metastasis largely contribute to this poor survival rate of HNSCC. Despite multimodality of treatments, HNSCC patients present with metastatic disease at the time of first diagnosis, leading to a reduction in their overall survival rate. Therapeutic intervention blocking progression of metastasis is of prime importance to increase the survival of HNSCC patients. The link between wound healing and cancer dates back to 1980's when solid cancers were defined as “wounds that do not heal”.¹ Cell migration, is a common event in wound healing and cancer: in the later, it is more complicated including trans-endothelial migration of cancer cells into vessels known as intravasation, followed by penetration of the endothelium and basement membrane; the process known as extravasation.²

In an attempt to understand the underlying molecular mechanisms that control cell migration and regulate normal wound re-epithelialization, we discovered a dual-state molecular switch which co-ordinates wound re-epithelialization and is essential for normal wound healing. The bi-stable switch, which is regulated by TGF-β controls context-specific expression of two alternate gene products from the same transcript. In normal skin, expression of anti-migratory microRNA-198 (miR-198) from the 3’-untranslated region of follistatin-like-1 (FSTL1) mRNA, switches to pro-migratory FSTL1 protein expression upon wounding. Temporal cell migration is enhanced, facilitating normal wound re-epithelialization.³ At this point, we speculated that carcinoma cells may hijack the wound-healing switch to enhance migration and metastasis.

Investigation of the wound healing switch in HNSCC revealed a defect in the switch, which shuts off miR-198 expression in favour of sustained FSTL1 production. A prolonged ‘wound healing’ phase is maintained which contributes to uncontrolled cell migration and metastasis. Epidermal growth factor (EGF) induces the expression of microRNA-181a (miR-181a), which downregulates the expression of its target gene KH-type splicing regulatory protein (KSRP).⁴ We know miR-198 belongs to a cohort of miRNAs which require KSRP for processing.⁵ In the absence or very low levels of KSRP, processing of miR-198 fails, the transcript now behaves as an mRNA and encodes the protein FSTL1. In summary, an EGF-driven micro-circuitry hijacks the molecular switch leading to sustained expression of pro-migratory FSTL1 and blocks miR-198 expression. In the absence of anti-migratory miR-198, its pro-migratory target gene Diaphanous 1 (DIAPH1), is significantly expressed in HNSCC. The two pro-migratory genes, FSTL1 and DIAPH1 promote EMT phenotype and promote metastasis. Overall we demonstrate how cancer cells manipulate the signalling pathway and regulatory switches crucial for normal physiological functions to enhance metastasis.

Using high throughput proteome profiling for interacting proteins, we identified Actin related protein 2/3 inhibitor (Arpin) as a specific binding partner for DIAPH1. Arpin was initially identified as a specific inhibitor of Arp2/3 complex, which generates branched actin filaments from pre-existing mother filaments, a process critical for lamellipodia formation and cell migration.⁶ In SCC, DIAPH1 competitively interacts and sequesters Arpin, preventing its interaction with Arp2/3 complex, to promote cell migration through lamellipodia.

On the other hand, the secretary nature of FSTL1, led us to focus on interacting partners that are extracellular in nature. Although, Wnt signaling has been characterized as tumor promoting pathway, Wnt7a is a tumor suppressor in lung cancer, due to its ability to inhibit EGF signaling.⁷ In SCC, FSTL1 restrains Wnt7a to promote expression Matrix metalloproteinase 9 (MMP9) by enhancing ERK phosphorylation. This creates a feedback loop whereby FSTL1, which is induced by EGF, enhances EGF signaling by activating MMP9, a protease required for extracellular matrix degradation.⁸

EGF/EGFR cascade is a classic example of a growth factor signaling that positions itself at the cross roads of wound healing and epithelial cancers. EGF stimulates keratinocyte migration and is required for the re-epithelialization phase in wound healing. On the other hand, over expression of EGF ligands or receptors are profoundly seen in head and neck cancer which significantly impacts the tumor growth and metastasis. This has led to the development of targeted approaches against EGFR or its downstream pathways in the form of monoclonal antibodies or tyrosine kinase inhibitors which have shown moderate success in clinic.⁹ It is worth noting here, despite the critical role of transforming growth factor β (TGFβ) in augmenting keratinocyte migration and wound healing, TGFβ signaling is often utilized in a contextual manner in cancers.¹⁰ Lack of TGFβ expression in our HNSCC sections, raises a possibility whereby HNSCC uses a failsafe mechanism to trigger this molecular switch by using EGF instead of TGFβ signaling.

The acquisition of the molecular phenotypes of wound healing in cancer is currently gaining considerable attention, especially in finding therapeutic targets. In the case of miR-198-FSTL1/DIAPH1 switch, it is apparent that by hijacking a single molecular switch, carcinoma cells acquire dual roles: first, shuts down a tumor suppressor microRNA-198 and second, enhanced expression two pro-oncogenic factors FSTL1 and DIAPH1 (Fig. 1). FSTL1-DIAPH1 synergistic gene-pair drives disease progression and metastasis, by two distinct pathways. Therapeutic intervention of defective molecular switch may lead to a novel strategy to effectively treat HNSCC, with better patient outcome.

Figure 1.

: Schematic representation of the molecular pathways, whereby the defective switch enhances metastasis.

Declaration

The authors declare no conflicts of interests and no competing financial interests.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Medical Research Council (NMRC) Individual Research Grant (IRG) to PS and Biomedical Research Council of Singapore, A*STAR.