Material Stiffness Directs Oral Cancer Migration - Project Summary Oral cancers of the mouth and/or pharynx are a common epithelial cancer with nearly 50,000 new cases/year in the US18 (affecting 3X more men than women19). Unlike other cancers, its rate of occurrence has increased 15% since the mid-1970s to be the sixth most common solid cancer. While 5-year survival rates for oral cancers have increased20, significant morbidity remains for common surgical treatments. Clinically, oral squamous cell carcinomas (OSCC) present as a stiff, necrotic lesion21 on the tongue and lips. Stiffened tissue–discernable by manual palpation–is a hallmark of most epithelial cancers, e.g., breast; stiffness is associated with extracellular matrix crosslinking and induction of epithelial-to-mesenchymal transition (EMT)1-4 via mechanically-activated pathways2,8. Our understanding of the role that stiffness plays in EMT for OSCC, and the pathways involved, however, is virtually non-existent. Preliminary data demonstrate that a stiffer niche converts oral epithelial cells into more aggressive mesenchymal-like cells of both dysplastic and invasive OSCC9,10. Similar to mammary epithelia in 2D5,6, these pre-malignant cells exhibit “memory” of their prior niche, i.e., cells cultured on stiff matrix recover prior behavior acquired in that past niche9,10 even once the cells have invaded adjacent tissue. Moreover, collagen expression and organization–indicators of tissue stiffness–is increased in patients with advanced disease and correlated with early recurrence9,10. Thus, we hypothesize that tumor stiffness creates new phenotypes that are consolidated and recovered via mechanical memory as OSCC cells invade new tissue and disseminate. We will use in vitro studies, in vivo mouse studies, and human OSCC samples to dissect the mechanisms that drive acquisition, consolidation, and recovery of mechanical “memories”. Aim 1: Elucidate the mechanisms involved in mechanical sensing leading to “memory” acquisition and recall. Dysplastic cells on a stiffer matrix have a more aggressive behavior22 resembling mesenchymal cells9. We hypothesize that EMT and memory acquisition involves crosstalk between cell adhesion, contractility and AKT phosphorylation whereas recovery of learned “memories” involves activation of migration-mediated signaling pathways, e.g., FAK. Dysplastic oral epithelial cells from patients and mice will be used to test this hypothesis with validation via patient histology. (A) We will analyze patient-derived cells for acquisition of EMT markers over time on stiff substrates. (B) We will then soften the niche to determine to what extent the acquired EMT markers become cell autonomous, i.e., they “learn” (via AKT) and “recall” EMT behaviors (via FAK). We will further assess how softening changes the transcriptome, adhesion23,24, migration, and invasion. Target inhibitors and activators as well as the FDA-approved OSCC drug, i.e., cetuximab–which affects migration25–will be assessed in vitro. (C) For human validation, OSCCs will be stained for markers of potential memory acquisition (e.g., AKT26) and recall (e.g., FAK) and patterns correlated to tumor region and patient prognosis. (D) For functional validation, we will use a carcinogen-induced mouse model (4-NQO)17 to determine if “learn” and “recall” inhibitors impact cell dissemination into tumor adjacent epithelia. (E) To determine if “memories” can be transferred via extrinsic signaling. We will culture naïve cells in media from or co-cultured with preconditioned dysplastic cells. Memory transfer to naïve cells will be assessed. Aim 2: Determine to what extent epigenetic changes in OSCC induce “memory” consolidation. Locally invasive, dysplastic cells encounter a soft niche in non-keratinized oral tissues27-29, so we hypothesize that stiffness-mediated “memory” consolidation is required via changes in histone acetylation and methylation prior to soft tissue invasion. We will determine to what extent epigenetics drive memory consolidation. (A) W
