(E-171) Ascitic shear stress promotes high grade serous ovarian cancer cell metastasis by activating NFkB and HSP27 via p38 MAPK signaling pathways

Introduction
Ovarian cancer is the 5th leading cause of cancer-related deaths among women worldwide and the most fatal of all gynecological malignancies, with a five-year survival rate of fifty percent [1]. Rapid disease progression is attributed in part to a dynamic microenvironment where ascitic currents both promote transcoelomic metastasis and stimulate tumors with elevated levels of fluid shear stress (FSS) [2]. FSS less than 0.1 dynes/cm2 have been shown to induce epithelial-to-mesenchymal transition (EMT) in high grade serous ovarian cancer (HGSC) [3,4]. Ascites volume also correlates with reduced survival, in which volumes greater than 1.8 liters lead to a 29-month decrease in median survival [5]. Moreover, ascites contains pro-tumoral soluble factors and tumor-associated macrophages (TAM) that promote metastasis and immunosuppression [6]. Ascitic currents arise from normal physiology such as breathing, digestion, and muscular-skeletal movement, however, no measurements of ascitic velocity currently exist. Though evidence has begun to support the influence of FSS on enhanced cancer stemness and metastatic potential, research has yet to investigate the influence of elevated FSS on epithelial-to-mesenchymal transition (EMT) and modalities for therapeutic intervention. To address this gap in knowledge, we have modeled ascitic currents from patient radiological data in silica and constructed a 3D perfusion bioreactor to apply these derived values of FSS to cells embedded within tumor-mimetic hydrogel. Our findings indicate that high HGSC cells exposed to our derived FSS undergo EMT by the mechanical modulation of mucin 15 (MUC15) and G-protein coupled receptors (GPCRs). Mucins play a variety of roles in epithelial tissue homeostasis including protection, barrier maintenance, and signal transduction. Transmembrane mucins, such as MUC15, are theorized to act as mechanical sensors and have been shown to aid in carcinogenesis [7].

Methods
Patient radiological data of ascites was used to model ascitic currents using a novel kinematic finite element analysis program, allowing the prediction of FSS on the ovarian surface. To investigate the effects of FSS on HGSC tumors, OVCAR3 and OVSAHO cells were suspended in an agarose-collagen I interpenetrating network hydrogel, with mechanical and transport properties comparable to the ovarian TME [8]. The embedded cells were then exposed to a pulsatile unidirectional FSS of 11 dynes/cm2 in a 3D perfusion bioreactor. After 24 hours, cells were recovered from the gel system and pathway analysis was performed by RNA sequencing, qPCR, western blot, immunofluorescent staining, and Ingenuity Pathway Analysis (IPA). Migration and invasion were evaluated by Boyden chamber after FSS exposure. The effect of MUC15 expression was investigated using OVCAR3 and OVCAR4 cell lines that were lentivirally transduced to either knockdown or overexpress MUC15. The migration and invasion were investigated by Boyden chamber. These cell lines were then exposed to FSS and evaluated by western blot and migration assay to determine the role of MUC15 in the FSS dependent cell behavior. To determine the immunomodulatory effects of the FSS induced soluble factors, the FSS conditioned media was recovered and analyzed by Multiplex Cytokine Array. Additionally, U937 human monocytes were differentiated into macrophages and co-cultured with OVCAR3 under FSS conditions and probed for M1 (CD86, HLA-DR) and M2 (CD206, CD163) macrophage polarization markers by flow cytometry.

Results, Conclusions, and Discussions
Stimulation of HGSC with physiologic FSS increased metastatic potential via induction of EMT, activation of p38 MAPK, and downregulation of MUC15. Computational modeling of ascitic currents based on diaphragmatic movement predicted an FSS greater than 5 dynes/cm2 on the ovarian surface. However, a maximum FSS of 11 dynes/cm2 is predicted when patient’s musculoskeletal movements are considered, these FSS are orders of magnitude larger than have been previously reported [9]. To understand the mechanism behind ascitic-mediated mechanotransduction, we applied a simulated predictive range of FSS, 1, 5, and 11 dynes/cm2, to OVCAR3 and OVSAHO representative HGSC cells, embedded within agarose/collagen type I hydrogel for 24 hours. FSS stimulation at 11 dynes/cm2 increased the cell motility and promoted more mesenchymal phenotype as evidenced by increased invasion and migration, increased actin cytoskeletal dynamics, and downregulation of E-cadherin (Fig. 1a). FSS stimulation contributed to increased cell motility by inducing phosphorylation of p38 MAPK and heat shock protein-27 (HSP27), an effector of F-actin trafficking. Consistent with the p38 MAPK activation, FSS increased nuclear localization of NF-κB, a transcription factor associated with immunosuppression and invasion. IPA based on RNA-seq indicated that FSS increased the secretion of cytokines such as IL-6 and TGF-β, which recruit TAM and promote an immunosuppressive TME, as confirmed from the conditioned medium from FSS-stimulated HGSC, and 3D hetero-spheroid model of HGSC-TAM [10]. Additionally, GPCRs were identified by IPA to be upstream of p38 MAPK activation, positioning GPCRs as a potential mechanosensor for FSS. We also found the expression of MUC15 to be significantly altered by FSS (Fig. 1b). To explore this further, we created MUC15KD using lentiviral transduced short hairpin systems and overexpressed lines in OVCAR3 and OVCAR4. Transcriptional modification of MUC15KD rescued FSS phenotype, enhancing cellular migration while overexpression displayed a modest decrease (Fig. 1c). Surprisingly, shear-mediated EMT does not appear to be dependent on the PI3K/Akt pathway as reported previously, but is instead reliant on the activation of p38 MAPK/HSP27 and NFκB. Replicating in vivo trends, our findings suggest a robust downregulation of MUC15 and the activation of EMT through p38 MAPK under ascitic FSS. By mechanistic investigation of MUC15, our study provides a fresh perspective on the glycocalyx in epithelial cells mechanosensing of the surrounding microenvironment. Overall, these findings solidify p38 MAPK and MUC15 as critical effectors of FSS-mediated HGSC progression and provide new avenues for targeted therapies.

Acknowledgements
This work is supported primarily by the American Cancer Society Research Scholar Award RSG-19-003-01-CCE (G.M.), NSF EFRI DChem (Award number 2029139). Research reported in this publication was supported by the National Cancer Institute under award number R37CA282790 and P30CA046592. We are grateful to undergraduate students (Alec R. Sunshine, Linh Tran, Zoe Jackson, Zachary Fisher, Liam Cotter) who participated in this research work.

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