Metabolomics, Transcriptomics and Functional Glycomics Reveals Bladder Cancer Cells Plasticity and Enhanced Aggressiveness Facing Hypoxia and Glucose Deprivation

Abstract

Bladder cancer constitutes one of the deadliest genitourinary diseases, especially when diagnosed at late stages. These tumours harbour microenvironmental niches characterized by low levels of oxygen (hypoxia) and limited glucose supply due to poor vascularization. However, the synergic contribution of these features to disease development is poorly understood. Here, we demonstrated that cells with distinct histopathological and molecular backgrounds responded similarly to such stimuli. Cancer cells arrested proliferation, significantly increased invasive capacity in vitro and enhanced tolerance to cisplatin-based chemotherapy. Reoxygenation and access to glucose restored basal proliferation and invasion levels without triggering stress-induced apoptosis, denoting significant cellular plasticity in adapting to microenvironmental cues. Whole transcriptomics showed major molecular reprogramming, supporting main functional alterations. Metabolomics evidenced fatty acids β-oxidation as main bioenergetic pathway rather than anaerobic glycolysis generally adopted by hypoxic cells. Joint pathway analysis also suggested relevant alterations in mucin-type O-glycan biosynthesis. Glycomics confirmed a major antagonization of O-glycosylation pathways, leading to simple cell glycophenotypes characterized by the accumulation of immature short-chain O-glycans such as Tn and STn antigens at the cell surface. Glycoengineered models reflecting simple cell glycophenotypes were developed and functional studies in vitro and in vivo showed that Tn and STn overexpression decreased proliferation and promoted chemoresistance, reinforcing their close link with tumour aggressiveness. Collectively, we have demonstrated that hypoxia and glucose deprivation trigger more aggressive cell behaviours, in what appears to be an escape mechanism from microenvironmental stress. We propose that, altered glycosylation may be used to target these subpopulations, paving the way for precision oncology.

Glycoproteomics identifies HOMER3 as a potentially targetable biomarker triggered by hypoxia and glucose deprivation in bladder cancer

“…We started by observing a striking decrease in T24 cells proliferation after deprivation of oxygen and glucose (Fig. 5), in agreement with our previous observations [18,45]. This was accompanied by an increase in cell invasion, which was tremendously amplified when normalizing the results to cell proliferation (Fig.…”

“…The presence of HOMER3 in each lane was estimated based on peptide-spectrum match (PSM), i.e. the total number of identified peptide spectra matched for the protein and T24 cells (data not shown), in accordance with our previous reports [18,45]. This demonstrated a metabolic reprogramming in response to the imposed microenvironmental challenge, as previously described by others [46][47][48][49].…”

“…5). These findings suggested that deprivation of oxygen and glucose acted as a selective pressure towards the selection/establishment of more aggressive and potentialy quiescent cancer cells, as widely described by different authors for different cancer models [45,[50][51][52]. Similar experiments conducted using HOMER KI, showed that HOMER3 upregulation induced proliferation in normoxia as well as in hypoxia in comparison to controls and wild type cells (Fig.…”

Glycoproteogenomics characterizes the CD44 splicing code driving bladder cancer invasion

“…7B). The link between STn and invasion is in close agreement with our previous report for glycoengineered BC cells (48), and other cancer models showing increased cell motility and acquisition of invasive traits resulting from the presence of this antigen (39,42,70). Nevertheless, subsequent studies showed that T24 C1GALT1KO and C1GALT1KO/ST6GALNAC1KI cells proliferation was not significantly affected by CD44s silencing (Fig.…”

“…Notably, positive PLA for CD44-STn was mainly observed in invasive fronts (Fig. 2C) consistent with the role played individually by CD44 and STn in cancer invasion (11,48).…”