PKIB facilitates bladder cancer proliferation and metastasis through mediation of HSP27 phosphorylation by PKA

Liu, Xiaolong; Yin, Xiaoyu; Yuan, Feng; Li, Shiqing; Xiao, Fei; Zhou, Feng; Pan, Xudong; Ho, Yatfaat; Dong, Shuo; Xu, Duan; Ma, Yunqing; Cao, Zhengding; Lei, Zhe; Sun, Yi

doi:10.1038/s41419-025-07814-7

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Published: 01 July 2025

PKIB facilitates bladder cancer proliferation and metastasis through mediation of HSP27 phosphorylation by PKA

Xiaolong Liu ORCID: orcid.org/0000-0001-7136-4299¹^na1,
Xiaoyu Yin²^na1,
Feng Yuan¹^na1,
Shiqing Li³^na1,
Fei Xiao⁴,
Feng Zhou³,
Xudong Pan⁵,
Yatfaat Ho ORCID: orcid.org/0000-0001-7478-7529⁶,
Shuo Dong²,
Duan Xu²,
Yunqing Ma⁴,
Zhengding Cao⁷,
Zhe Lei ORCID: orcid.org/0000-0002-5220-7391⁸ &
…
Yi Sun ORCID: orcid.org/0000-0002-2851-3401^2,9

Cell Death & Disease volume 16, Article number: 470 (2025) Cite this article

Subjects

Abstract

Cyclic AMP-dependent protein kinase A (PKA) is recognized for its pivotal involvement in various cancer types, with Protein Kinase Inhibitor Beta (PKIB) serving as an endogenous inhibitor that curtails PKA activity. Despite the documented escalation of PKIB expression in several malignancies, a comprehensive understanding of its precise mechanistic implications in human cancers remains elusive. This investigation is centered on bladder cancer (BLCA), unveiling an augmented expression of PKIB concomitant with heightened BLCA cell proliferation, migration, and invasion in vitro and augmented tumorigenic potential in an in vivo model. Mechanistically, PKIB disrupts PKA kinase activity, thereby resulting in diminished phosphorylation of the substrate target protein HSP27 at serine 15, 78, and 82. Additionally, the transcription factor MYCN exhibits an affinity for the PKIB promoter, leading to its enhanced expression in the context of BLCA. These findings reveal the oncogenic proclivity of PKIB and introduce a novel signalling pathway in BLCA, providing valuable insights into potential therapeutic targets for precise intervention.

Introduction

Cell growth is regulated by signal transduction pathways, and their dysregulation can contribute to cancer. Post-translational modifications such as phosphorylation, ubiquitination, acetylation, and methylation are key to controlling these pathways. Abnormal modifications can drive tumorigenesis by disrupting cell signaling [1]. Since kinase/phosphatase signaling dysregulation is common in tumors, pinpointing specific signaling interactions is crucial for developing targeted cancer therapies [2]. Cyclic AMP–dependent protein kinase A (PKA) is a versatile enzyme that plays a crucial role in various signaling pathways, influencing both normal physiological processes and disease states, including cancer [3]. PKA functions as a tetramer made up of two regulatory (R) and two catalytic (C) subunits. When cyclic AMP (cAMP) binds to the regulatory subunits, it triggers the release and activation of the catalytic subunits, which then phosphorylate serine and threonine residues on target proteins [4]. PKA has been demonstrated to promote cell migration and invasion, enhancing signal transduction and facilitating early cell cycle progression [5,6,7]. PKA has attracted extensive attention in cancer research [8]; however, its role in in tumorigenesis is complex and context-dependent, varying according to cancer types, stages, and other factors [9]. The cAMP/PKA signaling pathway is implicated in various facets of malignancy, including invasion, migration, adhesion, and clonal development, within a range of cancers encompassing glioblastoma, ovarian, colorectal, breast cancer, esophageal squamous cell carcinoma, and pituitary tumors [10,11,12,13,14,15]. Nevertheless, PKA has also demonstrated tumor-suppressive effects in certain cancers. Research indicates that PKA exhibits tumor-suppressive properties in glioblastoma cells [16,17,18]. In the context of lung cancer, PKA enhances apoptosis induced by radiotherapy by promoting phosphorylation of PP2A and upregulating AP1 expression [19, 20]. The spatiotemporal specificity of PKA is exquisitely controlled, particularly by the A-kinase anchoring proteins (AKAPs), the regulatory subunits of PKA, and the endogenously expressed PKA inhibitory peptides (PKIs) [21]. Nevertheless, our understanding of PKA signaling merely skims the surface of an extremely intricate phosphorylation network. The substrate specificity of PKA kinases is considerably more diverse than expected and is driven extensively by negative selectivity [22]. Consequently, targeting PKA signaling through specific inhibitors is essential for both cancer therapy and research endeavors. Such interventions could profoundly enhance our understanding of the intricate mechanisms governing downstream signaling pathways associated with PKA [23]. Despite the structural characterization of the PKA catalytic subunit being established for over three decades, the market for PKA inhibitors remains constrained, with no clinical approvals to date [24].

PKIB, denoted as cAMP-dependent protein kinase inhibitor β, is an integral part of the PKIs class, composed of endogenous peptides with the ability to suppress PKA kinase activities [25]. These inhibitors, including the α, β, and γ isomers, engage with PKA’s catalytic subunit via an unphosphorylated pseudosubstrate sequence at the N-terminus, consequently inhibiting PKA [26, 27]. PKIA (PKI-α) is a specific PKA inhibitor extensively researched for its binding and inhibitory capabilities [28]. Notably, PKIB (PKI-β) shares only 40% amino acid identity with PKIA and demonstrates more cell- and tissue-specific expression patterns [23]. Since its identification in rat testis in 1977, the majority of our current understanding regarding the function of PKIB is derived from its homologous protein PKIA; however, its exact functional role remains largely underexplored. A few studies have reported the abnormal overexpression of PKIB in a range of cancers, suggesting its potential role in tumorigenesis. In prostate cancer (PC), PKIB is notably overexpressed in castration-resistant and aggressive subtypes, where it enhances cell growth and invasion by increasing the kinase activity of PKA-C and the phosphorylation of AKT [29]. Similarly, in non-small cell lung cancer (NSCLC), PKIB is upregulated, leading to increased cell proliferation and tumorigenesis through the activation of the PI3K/AKT pathway [30]. PKIB also promotes the proliferation of highly metastatic osteosarcoma cells via the AKT pathway [31]. Additionally, elevated PKIB expression and its clinical significance have been identified in triple-negative breast cancer [32,33,34] and colorectal carcinoma [35]. Notably, a correlation has been observed between PKIB expression and AKT phosphorylation levels, as well as between AKT pathway activation and the effects of PKIB in these tumors. These evidences imply a significant potential oncogenic function of PKIB in multiple cancer types. Nevertheless, despite the aforementioned reports suggesting that PKIB might have a role in tumor progression by modifying the kinase activity of PKA-C and altering AKT phosphorylation, as of now, there is no proof that AKT is a substrate for PKA. The exact mechanism through which PKIB governs tumor progression remains unclear, especially the direct substrate of PKA kinase in tumor malignant progression mediated by PKIB and the overexpression regulation of PKIB in cancer cells, all of which demand further exploration.

Bladder cancer (BLCA) ranks as the most commonly diagnosed cancer of the urinary system. Recent trends indicate a gradual increase in both its incidence and mortality rates, with approximately 573,000 new cases and 213,000 deaths occurring annually [36]. BLCA is categorized based on the depth of invasion into two main types: muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). Among these subtypes, MIBC has a high degree of malignancy, strong invasive ability, and rapid clinical progression [37]. Despite recent advances in surgical treatment and chemical diagnosis, the effectiveness and specificity of treatments for BLCA remain limited, leading to frequent patient recurrence and disease progression [38]. The 5-year overall survival (OS) rate for patients with MIBC ranges from approximately 60% to 70%. Approximately 10% of patients with BLCA present with disease beyond the bladder, with an associated 5-year OS rate of 5–30% [39]. BLCA is the most underfunded of the most common cancers, which stifled research and contributed to a limited understanding of bladder tumor biology, ultimately led to insufficient progress in treatment [39]. Consequently, advancing research into the molecular mechanisms underlying bladder tumor development and malignant progression is essential. Such research is critical for the identification of novel therapeutic targets and the development of more effective diagnostic and therapeutic strategies for BLCA.

Current evidence suggests that PKA may play a pivotal role in the regulation of BLCA cell invasion, although the findings are currently sporadic. The PKA activator cholera toxin has been observed to specifically inhibit cell proliferation and induce G1 phase arrest in human bladder transitional cell carcinoma (TCC) cells, with these effects being reversed by the PKA inhibitor KT5720 [40]. Additionally, the cAMP/PKA signaling pathway has been shown to impede BLCA cell invasion through the modulation of MAP4-dependent microtubule dynamics [41]. There is evidence that PKA signaling may also mediate the anti-cancer effects of garlic extract in BLCA prevention [42]. Moreover, PKA inhibition has been linked to the attenuation of cAMP-induced reductions in BLCA cell mobility in animal models [43]. These results suggested that the inhibition of PKA kinase is strongly related to the pathogenesis and progression of BLCA. Given the role of PKIs in regulating PKA kinase activity, the study of PKIs may help to reveal the specific functional mechanisms of PKA in tumors. However, the function of PKIs (including PKIA, PKIB, and PKIC) in regulating BLCA progression is currently unclear.

In this study, we elucidated the critical roles and molecular mechanisms of the PKIB as a tumor promoter in BLCA. Our findings indicate that PKIB facilitates a reduction in the phosphorylation of the novel substrate protein HSP27 by inhibiting PKA kinase activity, thereby promoting tumor cell proliferation and migration. Furthermore, the transcription factor MYCN is implicated in regulating the overexpression of PKIB. These results illuminate the oncogenic role of PKIB and reveal an innovative signaling axis involving MYCN-PKIB-PKA-HSP27 in BLCA, enhancing our understanding of the intricate cellular pathways and regulatory mechanisms mediated by PKA kinases while providing valuable insights into PKIB as a potential novel therapeutic target for targeted intervention in bladder tumors.

Results

PKIB is highly expressed in BLCA, and its high expression is associated with BLCA progression

As the role of PKIB in tumors has not been determined, we investigated PKIB expression in public human cancer databases using Gene Expression Profiling Interaction Analysis 2 (http://gepia2.cancer-pku.cn). PKIB mRNA is overexpressed in 13 out of 31 cancer types, including bladder urothelial carcinoma, breast invasive carcinoma, glioblastoma multiforme, kidney chromophobe, prostate adenocarcinoma, etc. However, it is downregulated in two different tumors, acute myeloid leukemia and stomach adenocarcinoma (Fig. 1A), indicating that PKIB might perform different biological functions depending on the tumor background. We then focused on the expression of PKIB in BLCA tissues. It has been reported that endogenous PKA inhibitors (PKIs) contain PKIA, PKIB and PKIG [44]. To evaluate the possible roles of different PKIs in BLCA, we assessed the mRNA levels of the three genes in The Cancer Genome Atlas (TCGA) database (Fig. 1B) and 40 pairs of BLCA samples (Table S2). Intriguingly, only PKIB exhibited significant upregulation in both the TCGA database and our clinical specimens (Fig. 1C). Moreover, higher protein level of PKIB were detected in BLCA tissues than in normal tissues (Fig. 1D and Table S3). Kaplan-Meier Survival Analysis demonstrated a significant positive correlation between PKIB overexpression and reduced overall survival (OS) in patients with BLCA (Fig. 1E and Table S3).

**Fig. 1: *PKIB* expression is associated with BLCA progression.**

To determine the clinical significance of PKIB overexpression in BLCA, immunohistochemistry (IHC) was performed on a tissue array containing 22 normal bladder tissue samples, 28 low-grade BLCA tissue samples and 47 high-grade BLCA tissue samples (Table S4). As shown in Fig. 1F, the percentages of patients with strong and moderate PKIB expression in normal urothelial tissues, low-grade BLCA tissues and high-grade BLCA tissues were 22.7%, 49.9% and 66%, respectively. The expression of PKIB increased significantly (Chi-square test, P = 0.0036) with aggressiveness and was highest in high-grade BLCA tissues. Collectively, these results suggest that a substantial elevation in PKIB may significantly contribute to the progression of BLCA.

PKIB promotes BLCA cell viability and invasion

To explore the biological functions of PKIB in BLCA cells, stable PKIB-knockdown T24, 5637 and RT112 cell lines were generated using lentiviruses harboring short hairpin RNA (shRNA) targeting PKIB (shPKIB-1, shPKIB-2) or nontargeting control shRNA (shNC) (Fig. 2A, Fig. S1A). Following this, CCK-8 and colony formation assays were utilized to quantify the proliferation potential of the knockdown cells. The results showed that knocking down PKIB in T24, 5637 and RT112 cells significantly attenuated their proliferation (Fig. 2B, D and Fig. S1C, S1D). To investigate the role of PKIB in the cell cycle, we conducted flow cytometry analysis, which revealed that PKIB knockdown caused a disruption in cell cycle progression, specifically leading to arrest at the G0/G1 to S phase transition (Fig. 2C).

**Fig. 2: Knockdown of *PKIB* inhibits the proliferation, EMT, migration and invasion of BLCA cells.**

Metastasis to distant organs is an important feature of BLCA. T24, 5637 and RT112 cells with PKIB knockdown exhibited decreased expression of mesenchymal phenotypic markers (Fig. 2E, Fig. S1A), including Vimentin and N-cadherin. Immunofluorescence (IF) also confirmed that PKIB knockdown in T24 cells reduced mesenchymal and increased epithelial markers (Figs. 2F, G) which indicated PKIB expression is positively correlated with the epithelial–mesenchymal transition (EMT) in BLCA cells. To assess the involvement of PKIB in BLCA metastasis, we conducted wound healing and transwell assays. As shown in Figs. 2H, I and Fig. S1B, the number of migrated and invaded cells was markedly decreased in PKIB-knockdown T24, 5637 and RT112 cells.

To further elucidate the oncogenic role of PKIB, we developed T24 and 5637 cell clones with stable expression of exogenous PKIB (Fig. S2A). In contrast to the PKIB knockdown phenotype, the PKIB overexpression phenotype significantly promoted cell growth (Fig. S2B) and accelerated cell cycle progression (Fig. S2C) in BLCA cells. Furthermore, overexpression of PKIB also led to increased levels of Vimentin and N-cadherin (Fig. S2A) and enhanced cell migration and invasion (Fig. S2D). Taken together, these data indicate that PKIB promotes BLCA cell proliferation, EMT, migration and invasion in vitro.

PKIB inhibits PKA activity and mediates the nuclear translocation of PKA-Cα (catalytic subunit)

Although PKIB is a member of the PKI family, its inhibitory effect on PKA has not been fully characterized [29]. To assess the effect of PKIB on PKA, we analyzed mRNA and protein levels of the catalytic subunit PKA-Cα in PKIB-silenced T24 cells. qRT‒PCR and Western blot analysis revealed that PKIB knockdown did not affect the mRNA or protein levels of PKA-Cα (Fig. 3A). However, the subcellular localization of the PKA-Cα protein was altered upon PKIB knockdown, leading to an increase in the cytoplasmic PKA-Cα level and a decrease in the nuclear PKA-Cα level (Fig. 3B).

**Fig. 3: PKA directly interacts with HSP27 and phosphorylates HSP27 at serine 15, 78 and 82 in BLCA cells.**

To evaluate the effect of PKIB on PKA kinase activity, we transfected T24 cells with siPKIB or the PKIB overexpression plasmid and treated them with the PKA inhibitor H89. The ELISA assay indicated that PKIB overexpression led to a substantial decrease in PKA activity, comparable to the effect of H89. In contrast, PKIB knockdown caused a significant increase in PKA activity. This elevated PKA activity associated with PKIB knockdown was entirely reversed by H89 (Fig. 3C). These findings suggest that PKIB inhibits the kinase activity of PKA in BLCA cells.

PKIB mediates the phosphorylation of serine 15, 78 and 82 in HSP27 via PKA

Given that the important roles of PKA in different cancer types may be mediated by different substrate proteins, we conducted a phosphoproteomic analysis using PKIB-knockdown T24 cells to identify potential PKA substrates regulated by PKIB in BLCA. In total, we identified 37 significantly upregulated phosphorylated proteins, including the known PKA substrate GSK3β (Fig. S3B, Table S5), and 30 proteins with significantly downregulated phosphorylation. Among the top 10 proteins sequentially ranked according to the fold change value of upregulated phosphorylation, 6 proteins (FRYL, SYNRG, SRRM2, HSP27, RAPH1 and ERBIN) contained the canonical PKA phosphorylation motif RXXS, but only the phosphorylation of HSP27 has been widely reported in tumor biology. Therefore, among these proteins, heat shock protein 27 (HSP27/HSPB1) has attracted our special attention, although previous studies generally suggested that HSP27 may not be a genuine substrate for PKA [45]. HSP27 typically possesses three potential phosphorylation sites: Ser15 (S15), Ser78 (S78) and Ser82 (S82) [46]. We also conducted Western blot analysis on T24 and other BLCA cells (RT112 and 5637) with PKIB knockdown (Fig. 3P, S3C, S4D), which likewise revealed a significant elevation in HSP27 phosphorylation (S15, S78, and S82). These outcomes imply that HSP27 might serve as a potential PKA-interacting protein in BLCA.

The interaction between PKA and HSP27 was first detected by a GST pull-down assay in which the GST-HSP27 fusion protein or GST alone was incubated with purified FLAG-tagged PKA. The pull-down results showed that under our experimental conditions, FLAG-tagged PKA bound to GST-HSP27 but not to GST alone (Fig. 3D). We further performed Co-IP assays using exogenous proteins in HEK293T cells cotransfected with HA-HSP27 and FLAG-PKA plasmids (Fig. 3E) or endogenous proteins in T24 cells (Fig. 3F), confirming that PKA interacts with HSP27. Moreover, immunofluorescence staining revealed that PKA was localized in both the cytoplasm and the nucleus, whereas HSP27 was almost exclusively localized in the cytoplasm. These two proteins predominantly colocalized in the cytoplasm. When PKIB was knocked down, additional PKA translocated from the nucleus to the cytoplasm where it colocalized with HSP27 (Fig. 3G). Thus, these findings suggest that PKA directly interacts with HSP27 in BLCA cells.

HSP27 is comprised of a central α-crystallin ___domain (ACD) that is bordered by flexible N-terminal (NTD) and C-terminal (CTD) regions. To further explore the structural determinants of the interaction between HSP27 and PKA-Cα, three different truncations of HSP27 were generated (Fig. 3H, left). The different truncations were then cotransfected with FLAG-tagged PKA-Cα into HEK293T cells. Co-IP assays showed that PKA coprecipitated with the NTD-containing ___domain but not with the ACD or CTD of HSP27 (Fig. 3H, right), indicating that the NTD ___domain is crucial for the interaction between HSP27 and PKA. Interestingly, three phosphorylation sites in HSP27 (S15, S78, and S82) are located in the NTD ___domain, and sequence analysis also revealed that all of these sites correspond to the canonical PKA phosphorylation motif RXXS (Fig. 3I).

We also constructed protein homology models for human PRKACA (PKA-Cα) and HSP27 and performed molecular docking and molecular dynamics (MD) simulations of the complex to study its conformational dynamics (Fig. S4). The single-protein models of PRKACA and HSP27 were evaluated using PROCHECK (Fig. S4A). As shown in Fig. S4D, >97% of the regions were favorable for HSP27, and this value was 100% for the PRKACA model. The top three best scored poses of the complexes (complex 1, score 1833; complex 2, score 1525; and complex 3, score 1511) were further evaluated by CCharPPI (https://life.bsc.es/pid/ccharppi) [47]. Seven comprehensive scoring functions, namely, ZRANK, ROSETTADOCK, PYDOCK_TOT, FIREDOCK, AP_PISA, CP_PIE and SIPPER, were used to evaluate the rationality of the conformations of the three complexes (Fig. S4E). According to the evaluation results, complex 1 was the best composite model, and this model was used for further analysis (Fig. S4B). To determine the structural stability of the PRKACA/HSP27 complex, we performed an all-atom molecular dynamics simulation of the constructed complex. The root mean square deviation (RMSD) of the Cα atoms was used to assess the deviation of the structures from the initial coordinates (Fig. S4F). The system demonstrated convergence between 200 and 500 ns, with RMSD values stabilizing in the final 300 ns of the simulations. The conformational changes in the complex mainly occur in the loop region of HSP27. Interestingly, the conformation of the binding interface of the two proteins was relatively stable, and the relative positions and distances of S15, S78 and S82 to the ATP molecule remained constant ( ~ 6.0 Å, ~7.5 Å and ~8.5 Å, respectively) and stable during the 500 ns dynamics simulation (Fig. 3J). The results of molecular docking analysis and molecular dynamics simulation showed that HSP27 has a strong binding affinity for PRKACA, and the serine residues S15, S78 and S82 of HSP27 are the most likely phosphorylation sites.

We subsequently tested whether PKA phosphorylates these three sites on HSP27. Since elevated cAMP levels can lead to PKA activation, we treated T24 cells with forskolin, a pharmacological activator of adenylate cyclase. The results showed that HSP27 phosphorylation at S15, S78 and S82 was significantly increased (Fig. 3K). Similarly, when PKA-Cα was overexpressed in T24 cells, the phosphorylation of the three sites in HSP27 was also increased (Fig. 3L). To exclude the influence of other kinases in vivo, a kinase assay was performed in vitro using purified GST-tagged HSP27 and the PKA-Cα active protein (Fig. 3M). Western blotting also revealed that phosphorylation at these three sites in HSP27 was increased. These results indicate that the S15, S78 and S82 sites of the HSP27 protein can be phosphorylated by PKA in BLCA cells. To investigate whether there are other serine residues on the HSP27 protein that can be phosphorylated by PKA, we constructed four nonphosphorylated HSP27 mutants, namely, the Ala substitution Ser15 (S15A), Ser78 (S78A) and Ser82 (S82A) mutants, as well as the S15A/S78A/S82A triple mutant (3 A). Serine phosphorylation of HSP27 was then detected in vitro (Fig. 3N). The results showed that PKA-mediated serine phosphorylation of HSP27 was significantly reduced in all three single mutants, with the S15A mutant having a lower phosphorylation level than S78A or S82A. In addition, serine phosphorylation was almost undetectable in the HSP27-3A mutant. Furthermore, we detected the binding of four HSP27 mutants to PKA by Co-IP assays (Fig. 3O). Compared with that of wild-type HSP27, the binding of the S15A, S78A and S82A mutants to PKA was significantly weakened, whereas the binding of the HSP27-3A mutant to PKA was limited. These results strongly suggest that PKA binds and primarily phosphorylates the S15, S78, and S82 serine residues of HSP27. To further explore the association between PKIB and PKA-mediated phosphorylation of HSP27, we examined the phosphorylation of three serine residues in HSP27 after PKIB overexpression, PKIB silencing and H89 treatment in T24 cells (Fig. 3P). PKIB silencing increased the phosphorylation of HSP27 at S15, S78, and S82. As with H89 treatment, overexpression of PKIB significantly inhibited the phosphorylation of these three sites. In addition, H89 blocked the effect of PKIB silencing on the phosphorylation of these sites in HSP27. These findings suggested that PKIB affects the phosphorylation of HSP27 by inhibiting PKA kinase activity in BLCA cells.

HSP27 overphosphorylation inhibits BLCA cells viability and invasion

We have observed that PKIB downregulation led to reduced proliferation and invasion of BLCA cells, accompanied by increased phosphorylation of HSP27. To investigate whether elevated phosphorylation of HSP27 can induce cell phenotypes similar to those associated with PKIB downregulation, we overexpressed a mimicking phosphorylated triple mutant of HSP27 in T24 cells in which serine residues at positions 15, 78, and 82 were mutated to aspartic acid (HSP27-3D). As shown in Fig. 4A, the protein levels of the mesenchymal markers Vimentin and N-cadherin were lower in HSP27-3D cells than in HSP27-overexpressing and control cells, indicating that elevated phosphorylation of HSP27 is negatively associated with epithelial–mesenchymal transition in BLCA cells. Immunofluorescence (IF) also confirmed that HSP27-3D increased epithelial markers and reduced mesenchymal (Figs. 4C, D). Transwell assays were further conducted to evaluate cell migration and invasion (Fig. 4B). The results indicated a significant reduction in both migration and invasion capabilities in HSP27-3D cells, whereas HSP27-overexpressing cells showed no notable alterations in these processes. Additionally, the impact of HSP27 phosphorylation on T24 cell proliferation was assessed. The HSP27-3D variant caused cell cycle arrest at the G0/G1 to S phase transition (Fig. 4E) and significantly impaired colony formation and proliferation capacity, as evidenced by colony formation assays (Fig. 4F) and CCK-8 assays (Fig. 4G). Remarkably, the overexpression of HSP27-WT had no effect on these phenotypes. To validate these findings in a clinical context, bladder cancer patient samples in the TCGA database were divided into low and high HSP27 expression groups, and overall survival was analysed using the Kaplan‒Meier Plotter (http://kmplot.com/analysis/). The analysis demonstrated that elevated HSP27 expression did not correlate with adverse prognosis in BLCA patients (Fig. 4H), which corroborates the findings from the cell phenotyping experiments. Taken together, these results indicate that phosphorylation rather than overexpression of HSP27 inhibits BLCA cell viability and invasion, which is similar to the phenotypic changes observed in PKIB-knockdown cells.

**Fig. 4: HSP27 overphosphorylation inhibits the proliferation, EMT, migration and invasion of BLCA cells.**

To further confirm that PKIB regulates these biological processes by altering the phosphorylation of HSP27, we performed rescue experiments using T24 cells overexpressing PKIB and HSP27-3D. Introduction of HSP27-3D restored the increase in the expression of the N-cadherin and Vimentin proteins induced by PKIB overexpression (Fig. 4I). We then assessed the effect of overexpressing PKIB and HSP27-3D on cell phenotypes. According to the CCk8 assay, HSP27-3D effectively counteracted the effects of PKIB overexpression and blocked the proliferation of T24 cells (Fig. 4J). In addition, HSP27-3D successfully restored the increase in cell migration and invasion induced by PKIB overexpression (Fig. 4K). Collectively, the above results demonstrate that PKIB promotes cell proliferation and invasion in BLCA by mediating the inhibition of HSP27 phosphorylation.

S15-mediated phosphorylation of HSP27 is more important for inhibiting BLCA cell proliferation and metastasis

As shown above, PKIB-mediated phosphorylation of HSP27 at these three serine residues results in a tumor suppressor effect. To further investigate which specific site phosphorylation has the greatest impact on inhibiting the oncogenic potential of BLCA, we substituted each serine with aspartic acid and constructed three single mutants of HSP27 that mimic phosphorylation in T24 cells, named S15D, S78D and S82D (Fig. 5A). CCK8 assays indicated that HSP27-S15D most significantly suppressed the proliferative capability of BLCA cells, while S78D did not cause significant changes in the cell growth rate (Fig. 5B). Moreover, compared with S78D and S82D, HSP27-S15D led to a greater decrease in the expression of the mesenchymal markers N-cadherin and Vimentin (Fig. 5C) and inhibited cell migratory and invasive capabilities more effectively in T24 cells (Fig. 5D). These results imply that S15 phosphorylation of HSP27 is more important than S78 or S82 phosphorylation for inhibiting BLCA cell proliferation and metastasis. Additionally, immunohistochemical (IHC) staining analysis was conducted to determine the clinical significance of HSP27-S15 phosphorylation in different BLCA tissues (Table S6). HSP27-S15 was strongly and moderately phosphorylated in 91.7% of normal bladder tissues (n = 12), compared with 89.6% in low-grade (n = 29) and 52.7% in high-grade BLCA tissues (n = 38) (Fig. 5E), suggesting that the phosphorylation level of HSP27-S15 decreased significantly (Chi-square test, P < 0.001) with the progression of BLCA.

**Fig. 5: Overphosphorylation of HSP27 at residue 15 is more potent in BLCA cells inhibition.**

HSP27 phosphorylation inhibits the activation of the AKT/MMP2 signaling pathway

To investigate the downstream molecular pathways involved in HSP27 phosphorylation in BLCA progression, RNA-seq analysis was utilized to compare T24 cells with HSP27-phosphorylated (HSP27-3D) and HSP27-overexpressing (HSP27-WT) cells. This analysis revealed 98 downregulated and 112 upregulated genes at the mRNA level in HSP27-3D cells (Table S7). KEGG enrichment analysis identified the PI3K-AKT signaling pathway as the most prominently enriched among the downregulated genes in HSP27-3D cells (Fig. 5F). Notably, previous studies have reported the direct interaction between HSP27 and AKT [48]. Considering the pivotal role of AKT in the PI3K-AKT signaling pathway and the necessity of AKT-S473 phosphorylation for its activation, we examined the phosphorylation status of AKT-S473 in T24 cells. Phosphorylation of AKT in HSP27-3D cells was significantly lower than that in HSP27-WT cells (Fig. 5G). Because PKIB knockdown leads to enhanced phosphorylation of HSP27, we further used the GEO database (GSE13507) to screen for upregulated pathways in BLCA tissues with high PKIB expression. Interestingly, the PI3K-AKT signaling pathway was also identified in the enrichment list (Fig. 5H), consistent with the results of the RNA-seq analysis above. We also observed reduced phosphorylation of AKT-S473 in T24 cells after PKIB knockdown (Fig. 5I). These collective results strongly suggest that PKIB-mediated phosphorylation of HSP27 inhibits the activation of the PI3K-AKT signaling pathway in BLCA.

In addition, RNA-seq analysis also showed that the MMP2 gene was significantly downregulated (log2FC = –2.6) in HSP27-3D T24 cells (Table S7). MMP2, known to be regulated by the PI3K-AKT signaling pathway, is a key factor in tumor metastasis [49, 50]. We first assessed the protein expression of MMP2 in PKIB-knockdown T24 cells and found that the MMP2 protein level was significantly reduced (Fig. S5A). Subsequently, we overexpressed MMP2 in PKIB-knockdown T24 cells and performed CCK-8 and transwell assays. As expected, overexpression of MMP2 successfully rescued the proliferative and migratory abilities of T24 cells inhibited by PKIB knockdown (Fig. S5B, S5C), suggesting that MMP2 may be an important downstream protein for PKIB to influences the BLCA phenotypes.

MYCN regulates the transcription of PKIB in BLCA

To further investigate the regulatory effect of PKIB gene overexpression on BLCA, we utilized the TCGA database to analyse the transcription factors that were positively correlated with PKIB mRNA expression. A total of 31 potential candidates (r > 0.3) were identified (Table S8). To determine which transcription factor could bind to the PKIB promoter (positions –3000 to –1), we employed the multiple EM for motif elicitation (MEME) analysis tool. This analysis revealed seven putative transcription factors (Table S8). The expression levels of these transcription factors in BLCA were subsequently assessed using the TCGA database through the Gene Expression Profiling Interactive Analysis 2 platform (http://gepia2.cancer-pku.cn). Notably, only MYCN exhibited both a positive correlation with PKIB expression and significantly higher expression in BLCA tissues compared to normal tissues (Fig. 6A, B). In addition, Kaplan‒Meier survival analysis using the Kaplan‒Meier Plotter demonstrated a significant association between high MYCN expression and poor prognosis in BLCA patients (Fig. 6C).

**Fig. 6: MYCN directly interacts with the *PKIB* promoter in BLCA cells.**

We conducted experiments to verify the role of MYCN in regulating PKIB transcription in BLCA cells. First, the expression of the PKIB gene was measured in T24 cells with MYCN knockdown (siRNA), and the results showed that both the mRNA and protein expression of PKIB were significantly reduced (Fig. 6D), suggesting that PKIB expression may be regulated by the MYCN. Then, we used the JASPAR database (http://jaspar.genereg.net/) to search for potential MYCN binding sites (MBS) in the PKIB gene sequence (Fig. 6E). Sequence analysis revealed two putative binding sites located at positions –159 to –148 (MBS-1) in the PKIB promoter region and 424–435 (MBS-2) in the first intron (Fig. 6F).

To test whether MYCN regulates PKIB gene transcription by binding to these two prediction sites, we constructed a series of dual-luciferase reporter vectors containing PKIB promoter fragments, including the wild type and three mutants (Mut1, Mut2 and Mut1/Mut2), as shown in Fig. 6F (left panel). The construct was then cotransfected into 293 T cells with the MYCN overexpression plasmid. Luciferase activity was increased in the wild-type PKIB promoter but was significantly reduced in both the Mut1 and Mut2 mutant strains. Notably, the luciferase activity of the Mut1/Mut2 double mutant was almost completely eliminated (Fig. 6F, right panel). We also designed luciferase reporter vectors containing PKIB promoter fragments of different lengths (PKIB-WT, PKIB-2 and PKIB-3), as shown in Fig. 6G. Compared with that of the PKIB-WT vector containing all the predicted sites, the luciferase activity of PKIB-2 containing only MBS-1 was significantly lower, whereas the luciferase activity of PKIB-3 (positions -2000 to -500) without MBS-1 and MBS-2 was completely eliminated in 293 T cells. This finding is consistent with the results for the mutants, suggesting that the specific sites in the PKIB promoter region are critical for its transcriptional regulation by MYCN. To further validate the results of these luciferase assays and to confirm the direct interaction between MYCN and the PKIB promoter binding site, we also performed a chromatin immunoprecipitation (ChIP) assay in T24 cells. In this analysis, we observed significant enrichment of MBS-1 or MBS-2 in the DNA fragments immunoprecipitated by the MYCN antibody (Fig. 6H), indicating direct binding of MYCN to these sites. Taken together, these results suggest that MYCN binds directly to the promoter of PKIB and regulates its transcription in BLCA.

PKIB silencing or HSP27 phosphorylation can inhibit the metastasis and proliferation of BLCA cells in vivo

Furthermore, we used in vivo tumor models to assess the potential of targeting PKIB to suppress BLCA metastasis and proliferation. First, T24 cells (2 × 10⁶) with stable PKIB knockdown or a negative control vector were intravenously injected into BALB/c nude mice. After 8 weeks of inoculation, the mice were humanely killed and their lungs were surgically excised and examined for metastatic lung lesions (Fig. 7A). The lungs were stained with Bouin’s solution and hematoxylin and eosin (H&E), we observed that the mice injected with PKIB knockdown T24 cells developed fewer metastatic lung nodules compared to those injected with control cells (Figs. 7B, C). We also established a subcutaneous mouse model by subcutaneously injecting BALB/c nude mice with 5637 cells of stable PKIB knockdown (Fig. 7D). The tumor tissue volume in mice engrafted with PKIB-knockdown cells was considerably smaller than that in control mice (Figs. 7E, F). Indeed, the tumor growth rate was decreased by PKIB knockdown (Fig. 7G). This was further confirmed by immunohistochemical (IHC) staining of tumor tissues from sacrificed mice, which showed lower expression of Ki-67 in PKIB-deficient tumor tissues than in control tissues. Strikingly, IHC staining also indicated that PKIB knockdown markedly increased the expression of p-HSP27-S15, p-HSP27-S78 and p-HSP27-S82 in tumors (Figs. 7H, I), suggesting that PKIB mediates the phosphorylation of HSP27 in vivo. To investigate the inhibitory effects of HSP27 phosphorylation on BLCA metastasis, we also conducted a complementary in vivo experiment. The results demonstrated that HSP27 phosphorylation effectively suppressed tumor metastasis in animal models (Fig. S6), which is consistent with our proposed mechanistic hypothesis regarding its tumor-inhibitory function. Taken together, these findings are consistent with our in vitro results and further support the conclusion that PKIB mediates HSP27 phosphorylation and plays an important role in promoting BLCA metastasis and proliferation.

**Fig. 7: Knockdown of *PKIB* suppresses BLCA cell metastasis and proliferation in vivo.**

Gemcitabine inhibits the proliferation of BLCA cells via the MYCN-PKIB-HSP27 axis

Gemcitabine is a well-established chemotherapeutic agent used in clinical trials [51, 52]. It has been reported to activate the PKA signaling pathway [53] and induce HSP27 phosphorylation in pancreatic cancer [53, 54]. To investigate whether the anticancer effect of gemcitabine in BLCA involves the MYCN-PKIB-PKA-HSP27 axis, we detected the protein expression of MYCN, PKIB, and HSP27 in gemcitabine-treated BLCA cells. T24 and 5637 cells were first treated with 10 nM gemcitabine for 24 h. As expected, these treatments significantly inhibited the proliferative capacity of both types of BLCA cells and induced cell death (Fig. 7J). We then conducted time-dependent experiments on gemcitabine-treated T24 and 5637 cells to detect the protein levels of MYCN, PKIB, and p-HSP27. Interestingly, as the duration of drug exposure increased, there were notable changes in protein expression. In particular, the expression levels of the MYCN and PKIB proteins decreased over time, while those of p-HSP27-S15, p-HSP27-S78 and p-HSP27-S82 all increased gradually. Importantly, no changes in HSP27 protein expression were observed in the gemcitabine-treated cells (Fig. 7K). These results imply that the anticancer mechanism of gemcitabine may be mediated through the signaling axis involving MYCN-PKIB-PKA-HSP27 in BLCA.

In conclusion, our data suggest that high expression of PKIB, regulated by MYCN, mediates reduced HSP27 phosphorylation through inhibition of PKA kinase activity, thereby inducing a malignant phenotype in human bladder cancer (Fig. 7L).

Discussion

Increasing evidence suggests that dysregulation of kinase/phosphatase signaling is a frequent pathological characteristic of tumors. Dysregulated cAMP–PKA signaling is frequently regarded as crucial for mediating various physiological and pathological effects associated with malignant tumors [3, 9]. The results presented in this manuscript provide critical molecular and biological insights into the role of the PKIB, an endogenous PKA inhibitor, in tumors, as an oncogene and a driver of tumor cell proliferation and invasion in BLCA. Protein kinase inhibitors (PKIs) are important regulators of intracellular signaling pathways that control cell growth and division. These proteins typically bind to PKA via pseudosubstrate sequences and act as negative regulators of PKA kinase activity. Three PKIs (PKIA, PKIB and PKIG) have been discovered [44], among which the role of PKIA in PKA has been widely reported [28, 55]. Notably, the PKIs are endogenous, potent, heat-stable, and highly specific PKA inhibitors that effectively blocks PKA-mediated substrate phosphorylation, representing one of the most efficient strategies for PKA inhibition and a potential therapeutic target in diseases with aberrant PKA signaling.

Although PKIB exhibits only 40% amino acid homology with PKIA, its binding interactions and inhibitory effects on PKA remain inadequately characterized [28]. In our study, we found that the expression of only PKIB was significantly upregulated in BLCA, suggesting that PKIB plays an important role in bladder tumors. Although PKIB is abnormally overexpressed in various cancers and promotes tumorigenesis, the detailed molecular mechanisms by which PKIB regulates tumor progression are unclear. To the best of our knowledge, the molecular mechanism of PKIB in tumors is mostly understood in prostate cancer, where PKIB promotes the growth and invasion of prostate cancer cells by increasing the phosphorylation of AKT [29]. However, these findings imply that PKIB enhances the activity of PKA kinase in prostate cancer, which contradicts the role of PKIs as pseudosubstrates for PKA. More importantly, there are currently no reports indicating that AKT is a substrate of PKA. If PKIB enhances the phosphorylation of AKT via PKA, the substrate of PKIB-mediated PKA kinase has yet to be identified. The impact of PKA on cancer cell proliferation, invasion, and metastasis may be attributed to its interaction with multiple substrates. The diverse functions of PKA are likely due to its numerous targets, each contributing to its multifaceted roles in cancer progression. In our work, we found that PKIB plays an important oncogenic role in BLCA by inhibiting PKA kinase activity and reducing the phosphorylation of a new substrate target protein, HSP27, which confirms the biological function of PKIB as a specific PKA inhibitor. PKIB overexpression is a common feature in multiple malignancies, suggesting that this PKIB-PKA-HSP27 regulatory axis may have broader implications across different cancer types. On the other hand, our findings indicate that PKIB plays a role in the nuclear translocation of PKA-Cα, either by promoting its nuclear import or inhibiting its export, aligning with observations in prostate cancer [29]. Unlike PKIA, which promotes PKA-C nuclear export through binding with its pseudosubstrate motif [55], PKIB’s effect on PKA-C is comparatively less pronounced despite sharing the same motif [28]. The intracellular translocation of PKIB involved in PKA-C appears to be different from that of PKIA.

HSP27 is a small molecular chaperone whose activity is regulated through dynamic phosphorylation. In humans, HSP27 is phosphorylated at three primary sites: Ser15, Ser78, and Ser82 [46]. This phosphorylation is mediated by various kinases, reflecting a complex interplay that varies with cell type and signaling context [45]. Key kinases involved in HSP27 phosphorylation include MAPK-activated protein kinases (MK2, MK3, MK5) and Protein Kinase D (PKD) [56]. MK2 and MK5 are particularly notable for their role in directly phosphorylating HSP27, impacting its oligomerization and stress response functions [45]. Although Protein Kinase A (PKA) has been reported to phosphorylate HSP27 in some studies [57,58,59,60], this association is contentious and may be indirect [61,62,63,64], involving intermediary kinases such as MK2 or MK5 [45]. The consensus in the field suggests that while MK2, MK5, and PKD are crucial for HSP27 phosphorylation, the role of PKA remains ambiguous and is likely dependent on cellular context. In this study, however, through protein interaction analysis (including GST pull-down assay, Co-IP assay and immunofluorescence) and conformational dynamics study (molecular docking and molecular dynamics simulation), we demonstrated that HSP27 is a direct substrate of PKA kinase in BLCA cells. We determined that PKA binds to the NTD ___domain of HSP27 and phosphorylates its S15, S78, and S82 serine residues. In addition, phosphoproteomic analysis and PKIB knockdown or overexpression also confirmed that the phosphorylation of HSP27 by PKA is regulated by PKIB. To our knowledge, this is the first clear evidence that HSP27 is a direct substrate of PKA. Previous reports have shown that both phosphorylated and nonphosphorylated forms of HSP27 are predominantly localized in the cytoplasm [65], and our immunofluorescence staining demonstrated similar results. PKIB is expected to interact with PKA through two primary mechanisms: it both inhibits the kinase activity of the PKA catalytic subunit and modulates its nuclear localization. These mechanisms operate independently, and PKIB might employ both pathways to modulate HSP27 phosphorylation in BLCA.

The tumorigenic and metastatic roles of HSP27 have been well described [46]. HSP27 exists in two distinct forms, characterized by their phosphorylation and oligomerization states, each with specific functions and subcellular localizations. The phosphorylated monomeric HSP27 is primarily engaged in signaling pathways within the cytoplasm and nucleus, while the multimeric (nonphosphorylated) form is mainly involved in protein folding and preventing aggregation [66]. Increased expression and phosphorylation of HSP27 have been correlated with various tumor features, including disease progression, metastatic potential, therapeutic resistance, and adverse prognosis [67]. Although the relevance of HSP27 in tumor biology is undeniable, the evidence for its expression level and clinical significance in bladder tumors is still controversial [68]. The role of this molecule in BLCA has not been clearly elucidated. In this study, we found that phosphorylated HSP27 inhibited BLCA cell viability and invasion, whereas overexpression of HSP27 (nonphosphorylated) had no effect on these phenotypes. Moreover, phosphorylation at S15 of HSP27 plays a more important role in inhibiting BLCA cells than phosphorylation at S78 or S82. This finding is also in line with the results of PKA-mediated phosphorylation assays in HSP27 nonphosphorylated mutants, with the greatest reduction in phosphorylation levels occurring in the S15A mutant. In addition, HSP27-3D rescued the proliferation, migration and invasion phenotypes of BLCA cells enhanced by PKIB overexpression, suggesting that the carcinogenic effect of PKIB is mediated by altering HSP27 phosphorylation. Furthermore, phosphorylated HSP27 reduces the phosphorylation of AKT at S473 and inhibits its activity in BLCA, which is consistent with previously reported results showing a direct interaction between HSP27 and AKT [48]. Collectively, these data demonstrate that HSP27’s role in BLCA is phosphorylation-state-specific, with S15 phosphorylation being functionally dominant. Our findings reveal that PKIB-mediated inhibition of HSP27 phosphorylation represents a pivotal mechanism in BLCA progression, providing new insights into context-dependent PKA signaling regulation.

Although PKIB has been reported to be upregulated in a variety of cancers, the regulatory mechanisms leading to its abnormal overexpression are unknown. In this study, we screened MYCN, a transcription factor that is positively associated with PKIB expression in BLCA, and demonstrated that MYCN directly binds to two sites in the PKIB promoter region and promotes its transcription. MYCN is a pivotal oncogene functioning as a DNA-binding transcription factor that interacts with the bHLH ___domain of the E box promoter region to regulate gene expression [69]. Dysregulation of MYCN is implicated in various malignancies, influencing tumor initiation, progression, and therapeutic response [70, 71]. Evidence highlights MYCN’s role in neuroblastoma, where it modulates cell proliferation and apoptosis through its interaction with the transcriptional target NLRR1 [72]. In prostate cancer, MYCN enhances tumor progression by transcriptionally activating genes such as PARP1 and BRCA1 [73]. Additionally, MYCN has been shown to contribute to chemoresistance in non-small cell lung cancer (NSCLC) by binding to the HES1 promoter, which suppresses apoptotic pathways. Elevated MYCN levels are associated with advanced NSCLC stages and poorer patient survival [74]. Recent findings in BLCA also indicate that MYCN is upregulated and regulates cell proliferation through interaction with circLAMA3 [75]. Our study further demonstrates that MYCN upregulates PKIB promoter activity, underscoring its critical role as a regulator of PKIB in BLCA.

To date, the oncogenic molecular mechanism of PKIB in various cancers has not been determined. The results of our study suggest that MYCN promotes PKIB overexpression in BLCA, thereby inhibiting PKA activity and reducing HSP27 phosphorylation. This potentially represents a novel mechanism by which the MYCN-PKIB-PKA-HSP27 axis regulates BLCA tumorigenesis. Interestingly, we also found that the chemotherapeutic agent gemcitabine has a significant effect on this axis. After treating BLCA cells with gemcitabine, the expression of MYCN and PKIB decreased over time, while the expression of p-HSP27 increased gradually, consistent with the inhibitory effects of these protein expression changes on cancer cells. It has been reported that gemcitabine activates the PKA signaling pathway [53] and induces the phosphorylation of HSP27 [54] in pancreatic cancer. Additionally, gemcitabine mediates pancreatic cancer cell growth inhibition by activating the p38 MAPK-MAPKAP-2 pathway, which results in the phosphorylation of HSP27 [76]. In BLCA, the inhibition of HSP27 (OGX-427, an antisense oligonucleotide targeting HSP27) has been reported to enhance cell susceptibility to gemcitabine [77]. Gemcitabine is a cell cycle phase-specific chemotherapeutic agent (pyrimidine antimetabolite) whose mechanism of action involves killing cells undergoing deoxyribonucleic acid (DNA) synthesis (in the S phase), and it also impedes cell transition across the G1/S phase boundary [78]. Gemcitabine is the standard first-line therapy for BLCA; however, the prevalence of gemcitabine resistance significantly impacts long-term patient survival [79]. Over the years, resistance to gemcitabine in BLCA has been largely understudied. Therefore, developing a more complete understanding of gemcitabine and establishing a rational basis for the design of effective approaches for treating gemcitabine resistance remain major clinical challenges. The response of the MYCN, PKIB and p-HSP27 proteins to gemcitabine found in our study implies that the inhibitory effect of gemcitabine on BLCA may be mediated through the MYCN-PKIB-PKA-HSP27 axis, which may provide new implications for the clinical treatment of BLCA.

Recently, several comprehensive genomic analyses of bladder cancers have provided detailed characterization of genetic and epigenetic alterations in BLCA subtypes [80,81,82,83,84,85,86]. While classification systems differ in subtype nomenclature and numbers, luminal and basal subtypes consistently show the greatest separation, with distinct expression profiles, differentiation patterns, oncogenic mechanisms, tumor microenvironments, and associated histological and clinical features [87]. Although none of the current numerous molecular subtypes criteria for BLCA uses PKIB gene as a subtype-specific marker, we also conducted a preliminary study on the oncogenic role and molecular mechanism consistency of PKIB in the major BLCA subtypes. We performed bioinformatic analyses of PKIB gene expression in several transcriptome datasets (GSE87304 and GSE124305) with molecular subtype information [88, 89] and in the vast majority [25] of BLCA cell lines from DepMap (Broad Institute Cancer Dependency Map) [90]. The results showed that there was no significant difference in the expression of the gene among different molecular subtypes (Fig. S7A–D). We also selected eight commonly used BLCA cell lines among three major subtypes [90] (RT4, SW780 and RT112 belong to luminal subtype, SCaBER, HT1376 and 5637 belong to basal subtype, T24 and J82 belong to non-type or mix subtype) for qRT-PCR to further confirm the results. There was also no significant difference in PKIB expression among these subtypes (Fig. S7E, F). Furthermore, PKIB knockdown significantly inhibited EMT, migration, invasion, and proliferation of 5637 (basal subtype), T24 (mixed subtype), and RT112 (luminal subtype) cells (Fig. 2 and Fig. S1), suggesting that the oncogenic role of PKIB in different subtypes of BLCA cells is conserved. In addition, phosphorylation levels at S15, S78 and S82 of HSP27 were significantly elevated in T24, RT112 and 5637 cells with PKIB knockdown (Fig. 3P and Fig. S3C, D), implying that PKIB-mediated phosphorylation of downstream target HSP27 is consistent in BLCA. These results provide a preliminary indication that although there are some differences in clinical and histological behavior and markers of BLCA subtypes, PKIB expression, oncogenic role and targets are similar across the major subtypes (basal, luminal, and mixed), indicating the conserved nature of PKIB in BLCA.

The role of PKIB remains not fully elucidated. While prior researches have investigated PKI’s impacts on specific kinases such as protein kinase G (PKG) and protein kinase C (PKC) [91,92,93,94], the effects on a wider range of kinases have yet to be thoroughly explored. It remains uncertain whether PKIB exclusively inhibits PKA or if it also affects other enzymes or proteins. On the basis of our proteomic analysis of PKIB-knockdown T24 cells, we identified a number of significantly up- or downregulated proteins, the vast majority of which are not known direct substrates of PKA kinase. Does PKIB alter the phosphorylation of these proteins by affecting the activity of other kinases or by other regulatory means? On the other hand, although PKIA and PKIG show no significant overexpression in BLCA, their potential functional redundancy with PKIB is worth investigating, especially considering the link between PKA inhibition and BLCA progression [40,41,42,43].While no pharmacological PKIB inhibitors currently exist, microRNA-495 has been shown to induce PKIB silencing [95], providing a potential tool for probing PKIB interactions.

In summary, this study elucidates the oncogenic role of PKIB and uncovers a novel MYCN-PKIB-PKA-HSP27 signaling axis in bladder cancer (BLCA), advancing our understanding of PKA-mediated regulatory mechanisms in tumorigenesis. Furthermore, our findings highlight PKIB’s potential as a therapeutic target and its unique function as an endogenous PKA-specific inhibitor, offering valuable translational implications for cancer research and targeted therapy.

Material and methods

Cell culture and reagents

Human bladder cancer cell lines T24, 5637, RT112 and human embryonic kidney 293 T (HEK293T) cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences. Each cell line was then subjected to STR-based identification and was not contaminated by mycoplasma. All cell lines were used for less than half a year within 25 passages. HEK293T cells were grown in DMEM (Procell, PM150213), 5637 and RT112 cells were grown in RPMI 1640 (Procell, PM150110), T24 cells were grown in McCoy’s 5 A (Procell, PM150710). The medium was supplemented with 10% fetal bovine serum (FBS) (Gibco, 1099-141). The cells were cultured at 37 °C in a humidified 5% CO2 incubator.

Transfection and lentiviral infection

Following the manufacturer’s protocol, short interfering RNAs (siRNAs) or plasmids were transiently introduced into cells using Lipofectamine 3000 (Invitrogen), with subsequent analysis performed 48 h post-transfection. Primer sequences for plasmid construction and siRNA sequences are detailed in Supplementary Table 1. For stable gene overexpression or knockdown, cells were infected with lentivirus from GenePharma Biotech (Shanghai) for 72 h and then selected with 2 μg/ml puromycin to establish stable cell lines.

Western blot analysis

Cell protein was extracted using RIPA lysis (Beyotime Biotechnology, P0013B) supplemented with phosphatase inhibitor (Sangon Biotech, C500017) and protease inhibitor (Sangon Biotech, C600387) and was boiled with native sample loading buffer (Sangon Biotech, C506032) and DTT (Sangon Biotech, B645939) at 95 °C for 10 min. Then protein was subjected to electrophoresis with SDS-containing polyacrylamide gels, and was transferred to activated polyvinylidene fluoride (PVDF) membrane (Millipore). After 2 hours of blocking with 3% BSA, the membrane was incubated with the primary antibody overnight at 4 °C. TBST buffer was used for washing three times for 10 min each. The appropriate HRP-conjugated secondary antibody was selected for incubation for 1 hour at room temperature. Finally, using a chemiluminescent HRP substrate (Fdbioscience), the membrane was visualized with an enhanced chemiluminescence detection system. The following antibodies were used for Western blotting: rabbit anti-PKIB (Abcam, ab233521), anti-HSP27 (Abcam, ab109376), anti-HSP27 S15 (Abcam, ab76313), anti-HSP27 S78 (Abcam, ab32501), anti-HSP27 S82 (Abcam, ab155987), anti-MYCN (Abcam, ab227822), anti-HA (Abcam, ab236632), anti-FLAG (Abcam, ab205606), anti-PKA C-α (Cell Signaling Technology, #4782), anti-AKT S473 (Cell Signaling Technology, #4060), anti-AKT (Cell Signaling Technology, #4691), anti-Lamin B1 (Cell Signaling Technology, #13435), anti-β-actin (ABclonal, #AC004), anti-phospho-serine (Bioss, bs-11993R), mouse anti-N-cadherin (BD Biosciences, 610920), and anti-Vimentin (BD Biosciences, 550513).

RNA extraction and real-time quantitative reverse transcriptase PCR (qRT–PCR)

Total RNA was extracted from cells or tissues using TRIzol (Invitrogen, 10296010) and converted to cDNA using HiScript II Q RT SuperMix (Vazyme, R223-01). qRT‒PCR experiments were performed in triplicate. To calculate the relative expression of PKIA, PKIB, and PKIG, the data were analysed using the ΔΔCt method. For each gene, the mRNA expression was unified to that of β-actin.

Co-immunoprecipitation

The cells were cultured on 10-cm dishes and lysed in IP lysis buffer (Thermo Fisher Scientific, 87788) for 20 min. Fifteen percent of the lysates was reserved as the input control. The left lysates were mixed with antibodies against FLAG (Abcam, ab205606), HA (Abcam, ab236632), PKA (CST, #4782), HSP27 (Abcam, ab109376) or IgG (CST, #3900) and rotated overnight at 4 °C. To capture the immune complexes, protein A/G magnetic beads (Thermo Fisher Scientific, 88802) were used and rotated at room temperature for 1 h. After being washed 3 times, the protein was boiled with protein loading buffer at 95 °C for 10 min, separated from the magnetic beads and was subjected to further Western blot analysis.

CCK-8 assay

In total, 2000 cells were plated in 100 μl medium in a 96-well plate per well. After culturing the cells for 24, 48, 72, 96 and 120 h, 10 μl of CCK-8 solution (Vazyme, A311, China) was added to each well and incubated for 2 h, after which the absorbance was measured at 450 nm.

Transwell assay of migration and invasion

For the migration assay, 600 μl serum-free medium was first added under the bottom of chamber in a 24-well plate (Corning, 353504), and 40,000 cells in 200 μl medium supplemented with 25% FBS were added to the upper chamber of transwell inserts (Corning, 353097). After cultivation for 48 h, the cells on the membrane were washed twice with PBS buffer, fixed with methanol for 30 min and stained with crystal violet. Finally, the cells on the upper surface were removed, and the positively stained cells were observed under an inverted microscope. For the invasion assay, Matrigel Matrix (Corning, 354248) was initially added on the upper chamber and incubated at 37 °C for 30 min.

Cell cycle assay

After digestion with trypsin solution (Beyotime Biotechnology, C0207), the cells were washed twice with cold PBS buffer and fixed with 75% ethanol for 24 h at 4 °C. Subsequently, the cells were washed twice with PBS buffer again and incubated with propidium iodide (PI) (Beyotime Biotechnology, C1052) at 37 °C for 30 min. The cell cycle was detected by flow cytometry (Beckman Coulter).

Wound-healing assay

T24 and 5637 cell lines were seeded in 6-well plates until about 80–90% confluence. Then, a 100-μl pipette tip was used to make straight scratches. The cells were cultured in 0% serum medium for another 24 h. Images of cells were taken in three random microscopic fields at 0 h and 24 h.

Chromatin immunoprecipitation (ChIP)

EZ CHIP KIT 22 ASSAYS (Millipore, 17-371RF) was used for the ChIP assay according to the manufacturer’s instructions. Briefly, T24 cells were grown on 6-cm dishes and fixed with 1% formaldehyde for 10 min at room temperature. Glycine was then added to quench the unreacted formaldehyde. After being washed twice with PBS buffer and lysed with SDS buffer, the nuclear pellet was collected and sonicated to yield cross-linked DNA fragments 400–1000 bp in length. Chromatin was incubated with anti-MYCN antibody and Protein G magnetic beads overnight at 4 °C. Mouse IgG antibody (ab190475) was used as a negative control. The DNA- protein complexes were eluted and decrosslinked to collect ChIP DNA. The DNA was subjected to PCR amplification. The primers used for ChIP-PCR are listed in Supplementary Table 1.

GST pull-down assay

The GST-HSP27 fusion protein was induced in E. coli BL21 and purified using a GST 4FF chromatographic column (Sangon Biotech, C600912) according to the manufacturer’s instructions. FLAG-PKA fusion protein was expressed in 293 T cells and purified using anti-FLAG magnetic beads (Bimake, B26102). Then, the GST-HSP27 fusion protein or GST protein was incubated with anti-FLAG magnetic beads overnight at 4 °C. The GST fusion proteins were subjected to SDS‒PAGE and confirmed by Coomassie blue staining. The bound proteins were detected by Western blotting.

In vitro phosphorylation assay

In total, 80 μl recombinant GST-HSP27 product from E. coli BL21 was added with 0.3 μg purified FLAG-PKA Cα protein in kinase buffer (35 mM Tris-HCl, pH 7.5; 10 mM MgCl2; 0.5 mM EGTA; and 0.2 mM CaCl2) with 200 μM ATP for 30 min at 30 °C in a final volume of 100 μl. Proteins were subjected to Western blotting after the reactions were stopped at 99 °C for 5 min.

PKA kinase activity assay

For some experiments, T24 cells were pretreated with 10 μM H89 (Selleck, S1582) for 1 h. After being washed with PBS buffer for 2 times, the cells were lysed with RIPA buffer. According to the manufacturer’s instructions, an ELISA was used to measure PKA activity (Arbor Assays, k027-h1). The absorbance was measured at 450 nm. The PKA concentration (U/ml) was calculated with an internal calibration curve.

Immunofluorescence staining

T24 cells were cultured on round coverslips, fixed for 20 min with 4% paraformaldehyde, washed 3 times with PBS buffer and incubated with 3% BSA in PBST buffer for 1 h. Following incubation with rabbit anti-PKA C-alpha (Cell Signaling Technology, #4782) and mouse anti-HSP27 (Santa Cruz, sc-13132) antibodies overnight at 4 °C. After being washed, the cells were incubated with goat anti-rabbit IgG H&L (FITC) antibody (Abcam, ab6716) and goat anti-mouse IgG H&L (Cy3) antibody (Abcam, ab97035) at room temperature for 1.5 h. Cells nuclei were stained with DAPI (Beyotime Biotechnology, C1006). Finally, images were captured using a confocal laser microscope (Leica).

Colony formation assay

The treated bladder cancer cells were seeded in a 6-well plate (1500 cells per well) and cultivated for approximately 3 weeks. Cells were then fixed for 20 min with 1 ml methanol and stained for 30 min with crystal violet.

Dual-luciferase reporter assay

To determine the effect of MYCN on the luciferase activity of the PKIB promoter, the sequences of the PKIB promoter and MYCN were constructed and inserted into the pGL3-Luc reporter vector and pCDNA3.1-HA vector, respectively. The plasmids were then co-transfected into HEK293T cells for 48 h, and a Dual-Luciferase Reporter Assay System (Promega) was used for luciferase reporter assays. The relative luciferase activity was unified to the corresponding Renilla luciferase activity.

RNA-seq analysis

The mRNA expression profiles of HSP27-overexpressing and HSP27-overphosphorylating T24 cells were determined via transcriptome sequencing on an Illumina HiSeq platform (Origingen). Briefly, total RNA was extracted, quantified and qualified using TRIzol Reagent (Invitrogen), TBS380 PicoGreen (Invitrogen) and NanoDrop spectrophotometer (Thermo Fisher Scientific). Sequencing libraries were prepared with the TruSeq RNA Sample Preparation Kit and then were loaded onto an Illumina HiSeq instrument, and sequenced using 2 × 150 bp paired-end reads. HISAT2 software was used to map the reads to the human genome (GRCh38). Differentially expressed genes (DEGs) with a P value < 0.05 and a fold change (FC) > 2 were chosen for further investigation. The cells in each group were analysed in triplicate.

Phosphopeptide correlation profiling

Phosphopeptide correlation profiling was performed for three biological replicates at Shanghai GenePharma Biotech. Briefly, 100 μg of protein was combined with 15 μl of SDT buffer. Subsequently, DTT, the detergent and other low-molecular-weight components were removed by two rounds of ultrafiltration (Sartorius, 30 kD) using UA buffer. Next, 50 μl of iodoacetamide was added for 30 minutes in darkness. The membranes were washed three times with UA buffer and subsequently washed two times with TEAB buffer. The proteins were digested with 4 μg of trypsin (Promega) in TEAB buffer overnight at 37 °C. The peptide mixture (100 μg) was labeled with TMT reagent (Thermo Fisher Scientific). Subsequently, the labeled peptides were subjected to desalting using a C18 cartridge and was subjected to enrichment for phosphopeptides. The pooled TiO2 flow-through (FT) and wash fractions were combined, and further phosphopeptide enrichment was carried out. The eluents from both the TiO2 and Fe-NTA processes were dried and subsequently reconstituted in formic acid buffer. LC‒MS/MS analysis was conducted using a mass spectrometer coupled to an Easy nLC system (Thermo Fisher Scientific) for 4 h. Phosphopeptides exhibiting a fold change >1.2 and a P value (Student’s t test) <0.05 were considered differentially expressed phosphorylated peptides.

Structural modeling, molecular docking and molecular dynamics simulation

The protein homology models for human PRKACA (PKA-Cα) and HSP27 were constructed by using the MODELLER V9.19 [96] platform and templates derived from the protein data bank (www.rcsb.org) with PRKACA (4W5B) and HSP27 from their respective Homo 24-mer structure (6DV5, chain A), respectively, with very slow refinement. The quality of the modeled structure was evaluated by PROCHECK [97]. The PRKACA and HSP27 complex was docked using ZDOCK 2.3.2 [98, 99]. Protein complexes were ranked using a scoring function that included pairwise shape complementarity, desolvation and electrostatic terms (for quality scoring, see [100, 101]). The constructed complex was then subjected to MD simulations to study the conformational dynamics. The non-bonded interaction potential was terminated in a smooth and controlled manner at 10–12 Å. Furthermore, the Coulomb interactions were treated using the particle-mesh Ewald method [102]. The LINCS algorithm [103] was employed to impose constraints on the hydrogen-containing bonds. The minimized structure was then heated gradually from 0 to 303.15 K over 100 ps, followed by a two-step equilibration process (each 100 ps). The temperature was initially set at 303.15 K. Following this, the system was subjected to an equilibration process at 1 atm, utilizing the Parrinello-Rahman barostat [104]. The system was then subjected to 500 ns of MD production using the GPU-enabled version of the GROMACS software package (version 2021.4) [105] with the AMBER99SB-ILDN force field [106] with an integration step of 2 fs. The GROMACS analysis toolkit was employed to analyse MD trajectories, with root mean square deviations (RMSDs) subsequently calculated.

Bioinformatic analysis of public datasets

The TCGA_BLCA dataset containing 408 BLCA and 19 normal tissues from the Genomic Data Commons (GDC) database (https://gdc.cancer.gov/) was downloaded to detect the mRNA expression of PKIs. The GSE13507 dataset from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) was downloaded, and the KEGG Rest API (https://www.kegg.jp/kegg/rest/keggapi.html) was constructed with clusterProfiler (version 3.14.3) in the R language to screen for upregulated pathways in BLCA tissues overexpressing PKIB. Differentially expressed genes (DEGs) with an FDR < 0.25, an abs. log2FC > 1 and an adjusted P value < 0.01 were considered to be significantly different in the GEO database.

Animal experiments

All animal procedures were approved by the Animal Care and Use Committee of Soochow University. Male BALB/c nude mice, aged 4–6 weeks, were procured from the Laboratory Animal Center of Suzhou Medical College, Soochow University. To minimize potential bias, all animals were randomly allocated to experimental groups, and investigators remained blinded to group assignments throughout data collection and analysis. No sex-related differences were observed in any experimental outcomes. For the xenograft studies, sample sizes were determined based on established protocols from previous experiments rather than predetermined statistical methods.

For the in vivo metastasis assay, PKIB-silenced and control T24 cells (2×10⁶) in 0.15 ml PBS were injected intravenously into the tail vein of mice. For macroscopic analysis of metastatic nodules, mice were euthanised after 8 weeks of inoculation and their lungs were fixed in Bouin’s solution. Lung tissues were histologically examined using H&E staining for micrometastatic foci analysis.

For the in vivo proliferation assay, PKIB-silenced and control 5637 cells (3×10⁶) in 0.15 ml of PBS were injected subcutaneously into the axilla of the mice. Every four days, a vernier calliper was used to measure the tumor formed by the cells. Tumor volume (V) was estimated by measuring the longest diameter (L) and the shortest diameter (W) of the tumor. V = 0.5×W²×L.

Human BLCA tissue specimens

Fresh BLCA tumor and corresponding paracarcinoma tissue specimens were obtained after informed consent from 296 patients with BLCA authorized by the Second Affiliated Hospital of Soochow University (Suzhou, China) according to the principles of the Declaration of Helsinki. Detailed clinical information of the patients is listed in Tables S2, S3, S4, S6. None of patients received chemotherapy or radiotherapy before surgical operation. The study was approved by the Academic and Ethics Advisory Committee of the Second Affiliated Hospital of Soochow University (JD-LK-2019-104-01).

Tissue microarray (TMA) analysis

The TMA slides were systematically evaluated through dual-pathologist review, with all tissue cores (1.5 mm diameter, each representing an individual BLCA patient) being independently scored by two experienced pathologists. For both PKIB expression and HSP27-Ser15 phosphorylation status, immunohistochemical staining intensity was semi-quantitatively assessed using a four-tiered grading system: 0 (Negative): No detectable staining; 1 (Weak): Faint yellow cytoplasmic signal; 2 (Moderate): Distinct brownish-yellow coloration; 3 (Strong): Intense dark brown deposition.

Immunohistochemistry (IHC) analysis

Tissues from the indicated mice were isolated, rinsed in PBS, fixed in 10% neutral buffered formalin for 24 h, dehydrated, and embedded in paraffin. Tissue sections of 5 mm thickness were deparaffinized, rehydrated, and then treated for antigen retrieval. After blocking in protein blocking buffer, tissues were incubated with primary antibody overnight at 4 °C followed by incubation with biotinylated secondary antibody. Endogenous peroxidase was quenched in 3% H₂O₂ in water for 10 min at room temperature. Antibodies were visualized with avidin-biotin complex using diaminobenzidine as the chromogen.

Statistical analysis

All the data were analyzed using GraphPad Prism 5.02 software. For statistical significance between two groups, an unpaired Student’s t test was applied. Variance between groups was tested by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests. All the data in the graphs are presented as the means ± SDs from at least three independent experiments except where otherwise stated. P < 0.05 was considered to indicate statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Data availability

All data generated or analyzed during the course of this study are included in the published article information file, as well as in the supplementary information file.

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Acknowledgements

This work was supported by the Basic Research Program of Suzhou (SKY2023123, SSD2024040), Youth Excellence Projects of Suzhou Health (GSWS2020031), National Natural Science Foundation of China (82473045), Strengthening Health through Science and Education General Program of Suzhou (MSXM2024004), Boxi clinical research project in the First Affiliated Hospital of Soochow University (XLC2024020), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author information

These authors contributed equally: Xiaolong Liu, Xiaoyu Yin, Feng Yuan, Shiqing Li.

Authors and Affiliations

Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China
Xiaolong Liu & Feng Yuan
Department of Genetics, School of Basic Medical Sciences, Suzhou Medical College of Soochow University, Suzhou, 215123, China
Xiaoyu Yin, Shuo Dong, Duan Xu & Yi Sun
Department of Urology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China
Shiqing Li & Feng Zhou
Department of Bioinformatics and Computational Biology, School of Life Sciences, Suzhou Medical College of Soochow University, Suzhou, 215123, China
Fei Xiao & Yunqing Ma
Department of General Medicine, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China
Xudong Pan
Department of Urology, Suzhou BenQ Medical Center, Suzhou, 215000, China
Yatfaat Ho
Institute of Epidemiology & Health Care, Faculty of Population Health Sciences, University College London, London, WC1E 6BT, UK
Zhengding Cao
Department of Pathology, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China
Zhe Lei
Clinical Medicine Research Institute of Soochow University and Suzhou BenQ Medical Center, Suzhou Medical College of Soochow University, Suzhou, 215123, China
Yi Sun

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Contributions

YS and XL, as corresponding authors, conceptualized the project, supervised the experiments, and provided funding. XY, FY, SL, ZL, FX, FZ, XP, YH, SD, DX, YM, and ZC contributed significantly to the project through the design, data acquisition, analysis, and interpretation. The manuscript was prepared by YS and XY, with all authors participating in critical revisions for intellectual content. All authors have read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Xiaolong Liu, Zhe Lei or Yi Sun.

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Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

Human BLCA tissues were collected after informed consent was obtained from all patients. The study protocol was conducted in accordance with the Declaration of Helsinki and has been approved by the Academic and Ethics Advisory Committee of the Second Affiliated Hospital of Soochow University (Approval No. [JD-LK-2019-104-01]). Animal experiments were conducted according to guidelines approved by the Animal Care and Use Committee of Soochow University. All animal experiments were approved by the Animal Care and Use Committee of Soochow University.

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Supplementary information

Supplementary materials

Supplementary Table S5

Supplementary Table S7

Supplementary Table S8

Supplementary original Western blots

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Liu, X., Yin, X., Yuan, F. et al. PKIB facilitates bladder cancer proliferation and metastasis through mediation of HSP27 phosphorylation by PKA. Cell Death Dis 16, 470 (2025). https://doi.org/10.1038/s41419-025-07814-7

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Received: 26 November 2024
Revised: 23 May 2025
Accepted: 17 June 2025
Published: 01 July 2025
DOI: https://doi.org/10.1038/s41419-025-07814-7