Bladder cancer is usually multifocal with high recurrence and metastasis, and the EMT process has been comprehensively involved in bladder cancer progression [24, 25]. Nonmuscle invasive bladder cancer (NMIBC) accounts for more than 70% of newly diagnosed bladder cancers, and more than half of NMIBCs ultimately develop recurrence; approximately 15% of NMIBCs progress to muscle-invasive and/or metastatic diseases, which are highly connected with the EMT process [26, 27]. The multifocality of bladder cancer has been hypothesized to originate from intraluminal monoclonal expansion (clonal) [28, 29] or spontaneous transformation of multiple cells by virulent environmental agents (field effect) [30]. It has been characterized by early genetic instability or aberrantly epigenetic methylation [31–33]. Our findings implicated that exosomes function as potential environmental agents as the “field effect” to promote bladder cancer progression, which might lead to higher severity of multifocal tumors or more malignant behaviors of NMIBC cells.
The key regulators of the EMT process are E-cadherin, N-cadherin, vimentin, Snail, Slug, Twist, and Zeb-2, and most of them have been correlated with bladder cancer progression via either genetic or epigenetic regulation [34]. Currently, little is known about the association between the epigenetic regulation of exosome-transmitted lncRNAs in the EMT process and bladder cancer progression. In this study, we found that exosomes derived from high-grade bladder cancer cells indeed promoted the viability, motility, and clonogenicity of low-grade bladder cancer cells via enhancement of the EMT process. Furthermore, we identified two novel exosomal lncRNAs, LINC00960 and LINC02470, that both play pivotal roles in the EMT process and promote the aggressiveness of bladder cancer cells (Fig. 9A).
Exosomes are key elements that can facilitate intercellular communication and modulate tumor cells by influencing major cellular processes such as apoptosis, cell differentiation, angiogenesis and metastasis [35]. Bladder cancer cells have been reported to undergo EMT transformation after exposure to muscle-invasive bladder cancer exosomes [36]. Exosomes have also been reported to enhance cancer progression and recurrence in hepatocellular carcinoma via the MAPK/ERK signaling pathway [37]. Our results showed that low-grade bladder cancer cells (TSGH-8301) treated with high-grade bladder cancer cell-derived exosomes (T24-Exos or J82-Exos) had an increase in mesenchymal proteins (N-cadherin and vimentin) and a decrease in E-cadherin. T24-Exos or J82-Exos also significantly promoted the migratory and invasive abilities of recipient TSGH-8301 cells. The morphology of the recipient cells gradually changed into spindle-shaped mesenchymal-like cells after T24-Exos or J82-Exos treatment (Supplementary Fig. 1). Comparatively, TSGH-8301-Exos treatment did not change their behaviors and morphology in an autocrine manner. This suggests that exosomes derived from cells of different aggressiveness lead to different levels of aggressiveness-promoting effects in recipient bladder cancer cells.
The three most representative EMT upstream pathways, β-catenin/TCF signaling, Notch signaling and Smad2/3 signaling, were assessed to determine their involvement in exosome-induced EMT. Aberrant activation of β-catenin/TCF signaling is involved in a number of tumors, most notably colorectal carcinomas. TCF4 has been reported to transactivate Snail, Slug and Zeb1 and promote the EMT process [38, 39]. Notch1 signaling increases the DNA binding ability of NF-κB and thereby induces the expression of MMP9, which remodels the extracellular matrix and facilitates the extravasation of several cancer cells. Notch also stabilizes cytoplasmic β-catenin and activates other pathways, such as ERK and NF-κB, which induce the expression of Snail, Slug and LEF-1 transcription factors [40–42]. Smad complexes bind to regulatory elements and induce the transcription of key genes associated with EMT. Expression of activated Smad2 promotes mesenchymal spindle tumor cell invasion, which also regulates the expression of Snail, Slug and Twist to suppress the expression of E-cadherin [43–45]. Our results showed that β-catenin expression can be induced by treatment with each type of exosome, but its downstream transcription factors, especially TCF4, were significantly induced by T24-Exo treatment alone. The expression levels of Notch1, Notch4, Smad2 or Smad3 were all significantly upregulated by T24-Exos or J82-Exos treatment. T24-Exos or J82-Exos also induced the expression of the EMT-transcription factors Slug, Snail, Twist and Zeb2, which ultimately suppressed E-cadherin expression but promoted N-cadherin, vimentin, MMP2 and MMP9 expression in bladder cancer cells.
Several lncRNAs are involved in the EMT process and the degree of malignancy of tumors [46]. The main advantage of lncRNAs that make them suitable as cancer diagnostic and prognostic biomarkers is their high stability while circulating in body fluids, especially when they are encapsulated in exosomes or apoptotic bodies [47]. Several lncRNAs are associated with bladder cancer initiation and progression. LINC00958 has a regulatory role in bladder cancer progression, as its knockdown decreases cell viability, migration, and invasion [48]. LINC00355 is upregulated in bladder cancer samples and contributes to apoptosis inhibition, cell proliferation, and migration [49]. The urothelial carcinoma-associated (UCA1) lncRNA is also involved in cell proliferation, migration, invasiveness, and drug resistance of bladder cancer cells [50]. LINC00152 is highly expressed in bladder cancer and confers its carcinogenic effects by activating the Wnt/β-catenin signaling pathway [51]. Cooperatively, exosomes and their contained lncRNAs are regarded as components of cell signal transmission that modulate endogenous cellular microenvironments because exosomes are able to relocate functional lncRNAs between cells [52]. In this study, we identified two novel lncRNAs, LINC00960 and LINC02470, that were highly expressed in high-grade bladder cancer cells and their cell-derived exosomes. To our knowledge, this is the first study to address the function of LINC00960 and LINC02470, not only in intracellular regulation of malignancy traits but also in intercellular communication, illustrating their driving force in influencing the behaviors of bladder cancer cells. Our data demonstrated that siLINC00960-Exos or siLINC02470-Exos derived from T24 cells significantly inhibited proliferation, migration, invasion, and colony formation in recipient cells compared with their parental T24 exosomes. In addition, T24 siLINC00960-Exos treatment significantly reduced the migratory capability of recipient cells compared to that with siLINC02470-Exos treatment, which implied that LINC00960 and LINC02470 regulates the EMT process via different routes. Moreover, we found that T24 siLINC00960-Exos or siLINC02470-Exos treatment obviously reduced β-catenin signaling, Notch signaling and Smad2/3 signaling; notably, the major EMT transcription factors, Snail and Slug, were significantly reduced, which led to the downregulation of N-cadherin and vimentin [53].
In summary, we demonstrated that high-grade bladder cancer cell-derived exosomes promoted the malignant traits of low-grade bladder cancer cells. T24-Exos or J82-Exos-transmitted LINC00960 and LINC02470 induced the aggressiveness of bladder cancer cells via autocrine or paracrine effects. In contrast, T24 siLINC00960-Exos or siLINC02470-Exos attenuated these aggressive-promoting effects and inhibited the EMT process. Thus, T24-Exos or J82-Exos-transmitted LINC00960 and LINC02470 could be important aggressiveness-promoting factors in bladder cancer progression (Fig. 9B). And it also implicated that intercellular epigenetic regulations play critical roles during bladder cancer progression.