MYOSIN II ASSEMBLY REGULATION THROUGH THE MYOSIN PHOSPHATASE TARGETING SUBUNIT 1 DETERMINES CORTICAL MECHANICS FUNCTION ACCORDING TO MYOSIN ASSEMBLY AND MECHANORESPONSE POTENTIAL IN PANCREATIC CANCER CELLS

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers as the fourth leading cause of cancer related deaths with a 5-year survival rate of 10.8%. A critical need is to develop new PDAC-specific prognostics and targets. To this end, we are investigating the role of the myosin phosphatase targeting subunit 1 (MYPT1) in PDAC cell mechanics. MYPT1 was originally implicated in PDAC progression through a clinical trial where it was targeted by an antibody response to a whole tumor cell vaccine. Through this trial, MYPT1 expression was also found to be upregulated in patient PDAC cells, compared to normal ductal epithelium, and in established PDAC cell lines. MYPT1 is a component of the myosin phosphatase complex where it binds to the PP1β/δ catalytic phosphatase subunit and the phosphorylated regulatory light chain (RLC) of non-muscle myosin II (NMII). The MYPT1 complex facilitates RLC dephosphorylation, which leads to inactivation of NMII. NMII bipolar thick filaments are largely responsible for cell shape changes and have been linked to the altered cell mechanics associated with cancer transformation and metastasis. As a major regulator of NMII, we hypothesize MYPT1 has implications in cancer cell shape control. To elucidate MYPT1’s function in PDAC, I developed an in vitro model system composed of engineered cell lines using CRISPR-mediated MYPT1 knockout at each stage of PDAC transformation and progression. Each of the cell lines was then characterized for alterations in NMII-mediated cortical mechanics and oncogenic behavior. Additionally, performed an in vivo orthotopic xenograft model to assess the tumor formation, growth, and metastatic abilities of these lines. I found that removing MYPT1 from the cell increase NMIIC assembly and reduced its mechanoresponse ability. At the cellular and tissue levels, the alteration in myosin IIC function causes reduced migration and dissemination in vitro and reduced tumor formation, growth, and metastatic potential in vivo. I conclude that the upregulation of MYPT1 specifically lends cells an increased deformation ability through the regulation of NMIIC dynamic assembly and mechanoresponse potential. My dissertation work demonstrates that targeting NMIIC assembly and mechanoresponse is a viable strategy to reduce PDAC tumor burden and metastasis

Dental cell sheet biomimetic tooth bud model.

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation

Dental cell sheet biomimetic tooth bud model.

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation

Fulvestrant increases the susceptibility of enzalutamide-resistant prostate cancer cells to NK-mediated lysis

Background Enzalutamide, a next-generation antiandrogen agent, is approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). While enzalutamide has been shown to improve time to progression and extend overall survival in men with CRPC, the majority of patients ultimately develop resistance to treatment. Immunotherapy approaches have shown limited clinical benefit in this patient population; understanding resistance mechanisms could help develop novel and more effective treatments for CRPC. One of the mechanisms involved in tumor resistance to various therapeutics is tumor phenotypic plasticity, whereby carcinoma cells acquire mesenchymal features with or without the loss of classical epithelial characteristics. This work investigated a potential link between enzalutamide resistance, tumor phenotypic plasticity, and resistance to immune-mediated lysis in prostate cancer.Methods Models of prostate cancer resistant to enzalutamide were established by long-term exposure of human prostate cancer cell lines to the drug in culture. Tumor cells were evaluated for phenotypic features in vitro and in vivo, as well as for sensitivity to immune effector cell-mediated cytotoxicity.Results Resistance to enzalutamide was associated with gain of mesenchymal tumor features, upregulation of estrogen receptor expression, and significantly reduced tumor susceptibility to natural killer (NK)-mediated lysis, an effect that was associated with decreased tumor/NK cell conjugate formation with enzalutamide-resistant cells. Fulvestrant, a selective estrogen receptor degrader, restored the formation of target/NK cell conjugates and increased susceptibility to NK cell lysis in vitro. In vivo, fulvestrant demonstrated antitumor activity against enzalutamide-resistant cells, an effect that was associated with activation of NK cells.Conclusion NK cells are emerging as a promising therapeutic approach in prostate cancer. Modifying tumor plasticity via blockade of estrogen receptor with fulvestrant may offer an opportunity for immune intervention via NK cell-based approaches in enzalutamide-resistant CRPC