Known for a high rate of metastasis and muscle cell invasion, bladder cancer comprises some of the most malignant cancer subtypes. Current immunotherapy approaches fall short in treating patients because they do not successfully target cancer-specific biomarkers. That is why novel biomarker discovery is crucial to the development of effective therapies with minimal off-target effects.
Bladder cancer cell-membrane glycoproteome has become a major focus of immunotherapy research for several reasons. Not only is it easily accessible to therapeutic ligands, but it also tells us a lot about the behavior of cancer cells. The cell glycoproteome and the tumor microenvironment are in a bi-directional relation, where one easily adapts to changes in the other. In particular, the protein composition and distribution at the cell surface change as the disease progresses.
Changes in glycosylation patterns are commonly observed in cancer biomarker discovery. In particular, cell surfaces contain truncated glycans, where the glycosylation is prematurely terminated via sialylation. Research suggests that these alterations are part of an adaptation strategy in response to excessive tumor growth (1). In cases of oxygen shortage (hypoxia) and nutrient deprivation, due to poor vasculature in large tumors, such an adaptation often leads to migratory and metastatic behavior (2).
Despite the positive correlation between sialyl-Tn antigen expression and bladder cancer cell metastasis, the main surface proteins involved remain ambiguous. Through a combination of glycoproteomics, mass spectrometry, and immunohistochemistry, researchers from the Portuguese Institute of Oncology discovered HOMER3, an intracellular protein under normoxia, as a novel targetable glycoprotein (3). They also validated its relation to cell surface reprogramming, sialylated glycan expression in hypoxia, and cell invasion.
The researchers first constructed the O-glycomics profile of bladder cancer cell lines (T24 and 5637) to explore prevalent glycan structures on cell surfaces. To confirm these findings, they ran immunofluorescent microscopy and flow cytometry with Peanut Agglutinin (PNA) lectin (Vector Laboratories) to detect sialylated T antigens.
Using a bioinformatics workflow, the researchers identified 903 glycoproteins that expressed sialylated T antigens. Upon in silico scanning of their protein expression dataset from Oncomine, only 95 of these proteins were upregulated in muscle-invasive bladder cancer, narrowing down the results. An expression matrix was implemented to rank these glycoproteins according to their expression in bladder tumors and healthy tissues.
Of the top-ranking glycoproteins, the researchers chose to specifically focus on Homer protein homolog 3 (HOMER3), as its glycosylation and overexpression in bladder cancer had not previously been characterized.
The localization of HOMER3 in cancer cell lines was monitored before and after the cells were deprived of oxygen and nutrients. The changes in HOMER3 phenotypes and their impact on proliferation and cell invasion were then evaluated using gene-editing strategies.
Next, HOMER3-bearing bladder tumor samples were screened with PNA lectin and Vicia Villosa (VVA) lectin for the detection of short-chain sialylated O-glycans.
Finally, non-pathological samples from various healthy tissues were screened for the simultaneous expression of HOMER3 and the sialylated O-glycans to confirm the specificity of this expression pattern in bladder cancer.
The starting point of the study was the identification of O-glycans in invasive bladder cancer cell lines. Although ST antigens were the most abundant O-glycan at the cell membrane, significant amounts were also found in healthy urinary tract lining, which suggested that ST antigen expression could not be the sole indicator of a diseased state. In contrast, the expression of STn antigen differed significantly from healthy cell lines to mildly and highly invasive cell lines. These findings emphasized the importance of STn antigens in the biomarker discovery of advanced bladder cancer.
Discovering Targetable Biomarkers
The researchers used a bioinformatics workflow to scan gene expression datasets and find glycoproteins with O-glycosylation sites. Although the initial scanning suggested 903 possible glycoproteins, only 95 were upregulated in muscle-invasive bladder cancer.
The 95 glycoproteins were subjected to a ranking system depending on their overexpression in bladder cancer and absence in other human tissues. Strikingly, MUC16 and CD44—two proteins frequently studied in bladder cancer—did not score high as potential biomarkers. On the other hand, GLUT1 and HOMER3 topped the list.
Zooming In on HOMER3
The researchers focused their attention on HOMER3 because it, unlike GLUT1, which has strong associations with cellular reprogramming in hypoxia (4), had never before been implicated in altered glycosylation in bladder cancer.
Initially, flow cytometry on two aggressive bladder cancer cell lines revealed that HOMER3 was highly expressed inside the cell but rarely at the cell surface. According to western blot with PNA lectin, the small percentage of HOMER3 at the cell membrane consisted of high molecular weight glycoforms carrying short-chain O-glycans.
To investigate the factors triggering HOMER3 migration to the cell surface, researchers applied oxygen- and glucose-deficient conditions to the cancer cell lines. Although overall HOMER3 expression did not change, significant HOMER3 translocation to the cell surface was observed.
They determined that the impact of HOMER3 on tumor progression was highly dependent on the tumor microenvironment. In normoxia, this impact translated as increased cell proliferation; hypoxia and glucose deprivation inhibited cell proliferation but facilitated migration. As hypothesized, HOMER3 trafficking to the cell surface was found to be an adaptation strategy of the tumor in response to insufficient oxygen and nutrients, which, in turn, provides an escape mechanism for tumor cells in uncontrolled growth.
HOMER3 Glycoproteomics in Aggressive Bladder Tumors
A previous study investigated the abundant types of sialylated O-glycans in different bladder cancer disease stages. However, it was necessary to validate these glycosylation patterns in the context of HOMER3. Five muscle-invasive bladder tumor samples were used to explore the glycan structures associated with HOMER3.
Immunofluorescence microscopy images showed that HOMER3 and glycan structures were in close proximity, substantiating their colocalization at the cell membrane. To determine whether these glycans were neutral (T and Tn antigen) or sialylated (ST and STn antigens), the researchers used two lectins from Vector Laboratories. VVA lectin and PNA lectin aided in the detection of STn and ST antigens, respectively.
Although ST antigen was present in all tumors, its abundance declined with increased tumor aggressiveness, especially in hypoxic conditions. In contrast, STn antigen expression increased. Combining these findings, the researchers deduced that while ST antigen was involved in initial oncogenic differentiation of the bladder tissue, STn expression took over in more advanced tumors to support immune escape and metastasis of cells lacking oxygen and nutrients.
HOMER3-STn pairing in Healthy Human Tissue
The final goal was to determine possible O-glycosylated HOMER3 expression in healthy tissue. This was necessary to ensure precise targeting of aggressive cancer cells and identification of off-target sites. The screening of proteomics datasets from 20 different healthy tissues revealed HOMER3 in the kidney and respiratory tract, although detailed histological testing is needed for confirmation. Furthermore, the researchers could not detect HOMER3 glycosites presenting cell membrane glycans. Further analysis with immunohistochemistry confirmed that STn and HOMER3 were not co-expressed in healthy tissue cell surfaces, helping establish HOMER3-STn as a bladder cancer–specific biomarker for future targeted therapies.
Conclusion and Future Work
With the bioinformatics workflow used in this study, HOMER3 was identified as a top-ranked targetable glycoprotein in muscle-invasive bladder cancer. Although it has been previously associated with neuronal signaling (5) and T-cell activation (6), its involvement in altered glycosylation at the cancer cell surface had not been explored. This study demonstrated for the first time that enhanced HOMER3 sialylation at the cell surface is triggered by hypoxia and nutrient deprivation in large tumors. More importantly, HOMER3 abundance at the cell surface strongly correlates with bladder cancer cell invasion.
Despite these promising results, research on HOMER3 as a reliable biomarker is still in its infancy. Substantial future work is needed to identify the molecular pathways involved in HOMER3 accumulation at the cell surface. Future studies should also elaborate on the glycosylation patterns and HOMER3 glycosylation sites in much wider patient datasets. Finally, the off-target sites, such as kidneys, require a detailed analysis to fully account for the possible side effects of targeted therapies.