The regulation of cancer cell invasive behaviour by Chloride Interacellular Channel 3 (CLIC3) and Transglutaminasae 2

A study into the role of secreted CLIC3 in tumour cell invasion. The initiation and progression of cancers is thought to be linked to their relationship with a population of activated fibroblasts, which are associated with tumours. I have used an organotypic approach, in which plugs of collagen I are preconditioned with fibroblastic cells, to characterise the mechanisms through which carcinoma-associated fibroblasts (CAFs) influence the invasive behaviour of tumour cells. I have found that immortalised cancer-associated fibroblasts (iCAFs) support increased invasiveness of cancer cells, and that this is associated with the ability of CAFs to increase the fibrillar collagen content of the extracellular matrix (ECM). To gain mechanistic insight into this phenomenon, an in-depth SILAC-based mass proteomic analysis was conducted, which allowed quantitative comparison of the proteomes of iCAFs and immortalised normal fibroblast (iNFs) controls. Chloride Intracellular Channel Protein 3 (CLIC3) was one of the most significantly upregulated components of the iCAF proteome. Knockdown of CLIC3 in iCAFs reduced the ability of these cells to remodel the ECM and to support tumour cell invasion through organotypic plugs. A series of experiments, including proteomic analysis of cell culture medium that had been preconditioned by iCAFs, indicated that CLIC3 itself was a component of the iCAF secretome that was responsible for the ability of iCAFs to drive tumour cell invasiveness. Moreover, addition of soluble recombinant CLIC3 (rCLIC3) was sufficient to drive the extension of invasive pseudopods in cancer cell lines, and to promote disruption of the basement membrane in a 3D in vitro model of the ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition. My investigation into the mechanism through which extracellular CLIC3 drives tumour cell invasiveness led me to focus on the relationship between CLIC3 and the ECM modifying enzyme, transglutaminase-2 (TG2). Through this, I have found that TG2 physically associates with CLIC3 and that TG2 is necessary for CLIC3 to drive tumour cell invasiveness. These data identifying CLIC3 as a key pro-invasive factor, which is secreted by CAFs, provides an unprecedented mechanism through which the stroma may drive cancer progression.

Quantification of variable palmar ligaments around the triquetrum-hamate joint determined by lunate type

The ligaments of the wrist are highly variable and poorly described, which is more obvious on the ulnar side of the wrist. Previous studies highlighted the potential differences within the ligaments of the wrist but no consensus has been reached. Poor tissue description and inconsistent use of terminology hindered the reproducibility of the results. Improved understanding of the morphological variations between carpal bones may facilitate improved understanding of the ligamentous structure within the wrist. This study aims to identify the potential variations between carpal bones that could be used to separate palmar ligamentous patterns around the triquetrum-hamate joint into subgroups within the sample population. Investigations were performed following a detailed nomenclature and a clear definition of ligamentous structures to facilitate detailed description and reproducible results. Quantitative analyses were conducted using 3D modelling technique. Histological sections were then analysed to identify the structure of each ligamentous attachment. Variable patterns of ligamentous attachments were identified. Differences were not only obvious between samples but also between the right and left hands of the same person. These identifications suggested that the palmar ligamentous patterns around the triquetrum-hamate joint are best described as a spectrum with a higher affinity of the triquetrum-hamate-capitate ligament and the lunate-triquetrum ligament to be associated with type I lunate wrists on one extreme and type II lunate wrists with the palmar triquetrum-hamate ligament, triquetrum-hamate-capitate ligament and palmar radius-lunate-triquetrum ligament attachments at the other extreme. Histological analyses confirmed pervious established work regarding the mechanical role of ligaments in wrist joint biomechanics. Also, there were no significant differences between the quantitative data obtained from the Genelyn-embalmed and unembalmed specimens (p>0.05). The current study demonstrated variable ligamentous patterns that suggest different bone restraints and two different patterns of motion. These findings support previous suggestions regarding separating the midcarpal joint into two distinct functional types. Type I wrists were identified with ligamentous attachments that are suggestive of rotating/translating hamate whilst type II wrists identified with ligamentous attachments that are suggestive of flexing/extending hamate motion based upon the patterns of the ligamentous attachments in relation to the morphological features of the underlying lunate type of the wrist. This opens the horizon for particular consideration and/or modification of surgical procedures, which may enhance the clinical management of wrist dysfunction.

Unravelling metabolism of Leishmania by metabolomics

The leishmaniases are neglected tropical diseases with an urgent need for effective drugs. Better understanding of the metabolism of the causative parasites will hopefully lead to development of new compounds targeted at critical points of the parasite’s biochemical pathways. In my work I focused on the pentose phosphate pathway of Leishmania, specifically on transketolase, sugar utilisation, and comparison between insect and mammalian infective stages of the parasites. The pentose phosphate pathway (PPP) is the major cellular source of NADPH, an agent critical for oxidative stress defence. The PPP uses glucose, reduces the NADP+ cofactor and produces various sugar phosphates by mutual interconversions. One of the enzymes involved in this latter part is transketolase (TKT). A Leishmania mexicana cell line deleted in transketolase (Δtkt) was assessed regarding viability, sensitivity to a range of drugs, changes in metabolism, and infectivity. The Δtkt cell line had no obvious growth defect in the promastigote stage, but it was more sensitive to an oxidative stress inducing agent and most of the drugs tested. Most importantly, the Δtkt cells were not infective to mice, establishing TKT as a new potential drug target. Metabolomic analyses revealed multiple changes as a consequence of TKT deletion. Levels of the PPP intermediates upstream of TKT increased substantially, and were diverted into additional reactions. The perturbation triggered further changes in metabolism, resembling the ‘stringent metabolic response’ of amastigotes. The Δtkt cells consumed less glucose and glycolytic intermediates were decreased indicating a decrease in flux, and metabolic end products were diminished in production. The decrease in glycolysis was possibly caused by inhibition of fructose-1,6-bisphosphate aldolase by accumulation of the PPP intermediates 6-phosphogluconate and ribose 5-phosphate. The TCA cycle was fuelled by alternative carbon sources, most likely amino acids, instead of glucose. It remains unclear why deletion of TKT is lethal for amastigotes, increased sensitivity to oxidative stress or drop in mannogen levels may contribute, but no definite conclusions can be made. TKT localisation indicated interesting trends too. The WT enzyme is present in the cytosol and glycosomes, whereas a mutant version, truncated by ten amino acids, but retaining a C-terminal targeting sequence, localised solely to glycosomes. Surprisingly, cells expressing purely cytosolic or glycosomal TKT did not have different phenotypes regarding growth, oxidative stress sensitivity or any detected changes in metabolism. Hence, control of the subcellular localisation remains unclear as well as its function. However, these data are in agreement with the presumed semipermeable nature of the glycosome. Further, L. mexicana promastigote cultures were grown in media with different combinations of labelled glucose and ribose and their incorporation into metabolism was followed. Glucose was the preferred carbon source, but when not available, it could be fully replaced with ribose. I also compared metabolic profiles from splenic amastigotes, axenic amastigotes and promastigotes of L. donovani. Metabolomic analysis revealed a substantial drop in amino acids and other indications coherent with a stringent metabolic response in amastigotes. Despite some notable differences, axenic and splenic amastigotes demonstrated fairly similar results both regarding the total metabolic profile and specific metabolites of interest.