This study uncovers upregulation of an alternative form of TG2 in metastatic androgen insensitive PCa cell lines in hypoxic conditions and in PRAD biopsies, which as such is emerging as a potential new marker of prostate cancer. By dissecting cancer gene expression driven by this TG2 isoform, an unbiased new link between TG2 and MUC1 has been established and regulation of MUC1 via transcriptional repression of AR by TG2 revealed for the first time. This finding explains, expands and gives mechanistic value to previous observations, with the same PCa cell types, that NF-κB is capable of binding the androgen receptor (AR) promoter leading to gene silencing. We show here that the truncated form of TG2 is a key driver in AR suppression and results into MUC1 upregulation.
The link between androgen independency and prostate cancer aggressivity is well established[5–7]. In line with previous findings[27], this study has confirmed that an inverse correlation exists between AR expression and TG2 expression in metastatic prostate cancer cell lines. Metastatic androgen insensitive prostate cancer cell lines PC3 and DU145 displayed high TG2 upregulation, induced in conditions of HIF-1α expression, contrary to androgen sensitive metastatic cell line LNCaP and primary prostate cancer lines. Moreover, hypoxia induced the expression of the truncated TG2 isoform (TGM2_v2) that unlike the canonical long form (TGM2_v1) lacks the C-terminal regulatory peptide which controls TG2 transamidation via GTP binding[23, 47, 48]. Several studies have proposed that the up-regulation of TG2 in cancer is linked with unconventional activation of NF-κB[23, 49, 50], via degradation of IκBα in an IKK-independent way[23]. In particular, in prostate cancer, NF-κB is capable of binding the AR promoter leading to AR silencing[27]. To gain insights in gene regulation dependent on TG2 isoforms, the long canonical TG2 form and the truncated dysregulated TG2 form were added back to a number of androgen insensitive PC3 clonal cell lines with stable TG2KO. Here we have shown that knockout of TG2 in androgen insensitive PCa cells leads to restoration of AR expression, which can be repressed again by the add-back of TG2 isoforms, either the long or the truncated form of TG2 in a significant way. In the same way, when cancer-related transcriptome was investigated using digital transcriptomics, MUC1 expression was revealed to be dependent on TG2 expression, since TG2KO lowered MUC1 expression and the truncated TG2 isoform significantly restored wild type levels of MUC1 when transfected back in the TG2KO clones.
Other transcripts were also upregulated by TG2KO. Two encoding extracellular proteins ABI3BP (ABI Family Member 3 Binding Protein), involved with collagen ECM and cell adhesion and MGAT5 (Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase) a glycosyltransferase involved in the regulation of the glycoprotein oligosaccharides biosynthesis affecting cell migrations of cells. Others include, EIF4E2 (Eukaryotic Translation Initiation Factor 4E Family Member 2) and HSP90B1 of cytosolic location. However, MUC1 was the only transcript specifically upregulated by the aberrant TG2 isoform significantly.
This is the first time that a link between TG2 and MUC1 was established. MUC1 is an oncoprotein overexpressed in human prostate cancer the regulation of which is still being understood[28, 40, 51]. As a type I glycoprotein, MUC1 is a transmembrane member of the mucin family, a heterodimer consisting of a large N-terminal fragment (MUC1-N) and a C-terminal subunit (MUC1-C) and it is widely expressed on the apical surface of most epithelial tissues. In androgen-insensitive cell lines, PC3 and DU145 cells, MUC1 is highly expressed[28, 51]. On the other hand, androgen-sensitive cell lines including LNCaP, CWR22Rv1, MDA-PCa-2b do not express or have little MUC1, but express AR, suggestive of an inverse relationship between the two [28, 51]. Work by Kufe’s group has shown that transfection of AR reconstitutes low levels of MUC1 in PC3 and DU145 cells, due to AR occupying a consensus on the AR element of MUC1 promoter[51]. Here we have shown that TG2KO restores AR expression in PC3 cells and at the same time lowers MUC1 transcript. Therefore, TG2 participates in MUC1 regulation via transcriptional repression of AR. In particular, it is the aberrant form of TG2, the truncated one to be involved in this process. The TG2 role appears to depend on TG2 transamidation, since a well characterised TG2 inhibitor (ZDON) recapitulates AR expression and MUC1 downregulation, although at a lower significance level than seen via TG2 gene silencing/add back.
In a previous study in PC3 cells nuclear extracts, a complex between RelA (p65)/NF-κB heterodimer and TG2 was reported to colocalise in the nucleus implying a nuclear translocation on the TG2- NF-κB, leading to transcriptional repression of AR[27]. Here, we have observed a nuclear localisation of the aberrant form of TG2 (TGM2_v2) the expression of which regulates MUC1 in PC3 TG2KO cell lines. This important new finding suggests that the truncated form of TG2 which we have found expressed in PC3 and DU145 upon hypoxic stimulus may be involved in the TG2-mediated activation of NF-κB leading to AR repression and MUC1 overexpression, a pathway linked with prostate cancer cells aggressivity.
Mucin-1 is aberrantly expressed in a number of human cancer types, including breast, ovarian, bladder, pancreatic, lung and prostate with loss of polarity in cancer cells, and differentially glycosylated[41, 51], with reports of poor survival, and poor response to therapy. MUC1 extracellular domain was reported to contribute to a decrease in the immune response in breast cancer cells[52]. The cytoplasmic C-terminal subunit has been attributed to its oncoprotein properties via signal transduction roles[41, 53, 54]. We show clinical significance in this by the positive correlation found between TG2 and MUC1 expression in PCa using archived data for five independent clinical studies. A link between the aberrant TGM2 transcript and PRAD was also established for the first time in this study when PRAD and CIP cohorts were compared.
Phenotypically TG2 silencing in two androgen insensitive PCa cell lines, led to clonal cell lines with impaired cellular adhesion and cell migration and anchorage independent spheroid formation. Several lines of evidence have previously shown that TG2 plays a role promoting migratory ability of cancer cells including breast cancer, pancreatic cancer, epidermal and neuroblastoma cells[18, 55, 56]. In lung cancer, recent evidence suggests that TG2 promotes migration and invasion by positively regulating the activation of Rac[19]. All PC3 TG2KO clones displayed reduced cell migration post deletion of TG2 and the DU145 TG2KO clones displayed defects in spheroid formation, suggestive of a role of TG2 supporting migration and anchorage independency. In addition, inhibition of transamidating activity of TG2 pharmacologically showed reduced migration of metastatic PC3 cells suggesting that TG2 promotes cell migration through its crosslinking activity. We know that TG2 is present in the cytosol and in the nucleus[57, 58], but it is also released from cells via extracellular vesicles[35, 37, 38] and here we have shown for the first time that TG2 is prominent in small extracellular vesicles of PC3 cells. Interestingly, we have found that EV not only contain TG2 but also mucin isoforms in the membrane and intraluminal fractions that may be communicated from cell to cell. Thus, the externalisation of TG2, a leaderless protein, via small and large EV is consistent with our recent observations that EV are carriers of TG2 and concur to the accumulation of TG2 in the extracellular environment. As TG2 has a role in anchorage independency and cell migration in two metastatic prostate cancer cell lines (PC3 and DU145), it is linked with the invasive phenotype and its secretion via EV could be part of the mechanism.
The link between TG2 and MUC1 is novel. Very recently while this report was prepared Recktenwald reported that epidermal transglutaminase-3 is able to cross-link and hence stabilise the gel forming mucus Mucin 2 with relevance in colon protection from inflammatory processes such as colitis[59]. We have revealed for the first time that MUC1 is also a cross-linking substrate for TG2. Therefore, TG2 participates in the mechanism of MUC1 overexpression but MUC1 is also stabilised by the same TG2 at the surface of epithelial cells. It is tempting to speculate that TG2 could help stabilise a MUC1 barrier, potentially making prostate cancers cells immune resistant. A coating of human pancreatic ductal carcinoma (PDA) by other filamentous proteins such as a keratin 19-CXCL2 complex stabilised by TG2 was recently proposed to evade the immune attack[60].
In conclusion, this study has identified a novel pathway mediated by TG2 which may contribute to androgen insensitive PCa cells aggressivity and resistance via MUC1 upregulation and aberrant expression of truncated TG2 isoform. Understanding how MUC1 can be stabilised by TG2 and the relevance in cancer immune evasion will clarify the full significance of this new finding.