The Mucin-Alginate Interplay: Investigating the Rheological Impact of Alginates and Their Influence on Particle Mobility

Mucus is a hydrogel that covers epithelial cells and acts as an intermediary between the exterior and interior surfaces of the human body. It is complex and diverse, comprised of various compounds and serves as a selective barrier to pathogens, nutrients and administered substances. It is therefore important to circumvent or penetrate this barrier to increase drug bioavailability. The most important constituent of mucus is mucin glycoprotein that enables gel formation. Due to the molecular nature of mucin, it interacts with most substances including polyanionic biopolymers like alginates. Alginate molecules have been extensively used in pharmaceutical and medical industries as mediators because they possess suitable characteristics for a variety of biomedical applications. They have also been shown to influence mucus rheology, and could hold potential to alter other properties of mucus as well, such as particle mobility. In this thesis, the effect of different molecular weight alginates were investigated on the rheology of porcine gastric (PG) mucin, porcine small intestinal mucus (PSIM), porcine tracheobronchial mucus (PTBM) and bio-similar mucus. In addition, the effect of alginate on particle mobility in PG mucin samples were investigated using multiple particle tracking (MPT). The rheological effect of alginate G-block DPn 12, DPn 24, DPn 33 and alginate LFR 5/60 on PG mucin was inconclusive as the sample suffered from pH and mechanical instabilities. Particle mobility was greatest for G-block DPn 12 and G-block DPn 33 treated PG mucin at long time scales. In addition, their mean-square displacement (MSD) trajectories were more narrowly distributed than the other samples, which correlates to a higher degree of uniform pore sizes. The rheological behaviour of bio-similar mucus and PSIM after treatment of G-block DPn 12, G-block DPn 33 and LFR 5/60 did not coincide, despite bio-similar mucus being a model system for porcine intestinal mucus (PIM). This was ascribed interactions between alginate and polyacrylate in the bio-similar mucus causing phase separation. Alginate G-block DPn 12 weakened the PSIM and PTBM gel, while G-block DPn 33 and LFR 5/60 had minor strengthening effects on PSIM

ELF3, ELF5, EHF and SPDEF Transcription Factors in Tissue Homeostasis and Cancer

The epithelium-specific ETS (ESE) transcription factors (ELF3, ELF5, EHF and SPDEF) are defined by their highly conserved ETS DNA binding domain and predominant epithelial-specific expression profile. ESE transcription factors maintain normal cell homeostasis and differentiation of a number of epithelial tissues, and their genetic alteration and deregulated expression has been linked to the progression of several epithelial cancers. Herein we review the normal function of the ESE transcription factors, the mechanisms by which they are dysregulated in cancers, and the current evidence for their role in cancer progression. Finally, we discuss potential therapeutic strategies for targeting or reactivating these factors as a novel means of cancer treatment

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAF V600E colorectal cancer

Abstract Metastatic BRAF V600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAF V600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAF V600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAF V600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAF V600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAF V600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients

Genomic Profiling of Biliary Tract Cancer Cell Lines Reveals Molecular Subtypes and Actionable Drug Targets

Biliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking

Abstract Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology