Abstract

   Identifying novel therapeutic targets for cervical cancer is crucial
   for improving patient outcomes and reducing the global burden of this
   disease. Gap junction protein beta 5 (GJB5) is a member of the connexin
   family of proteins involved in cell-to-cell communication. This study
   investigated GJB5’s expression and functional significance in cervical
   cancer. Analysis of The Cancer Genome Atlas (TCGA) data demonstrated
   significantly increased GJB5 mRNA expression in cervical cancer tissues
   compared to normal cervical epithelium. Moreover, high GJB5 expression
   correlated with reduced overall survival and other adverse clinical
   outcomes. Single-cell RNA sequencing corroborated GJB5 overexpression
   within the malignant tumor cell population. The downregulation of GJB5
   through shRNA or CRISPR/Cas9 gene knockout techniques significantly
   impaired the viability, proliferation, and migratory capacity of
   cervical cancer cells, while concurrently inducing apoptotic processes.
   Conversely, the forced overexpression of GJB5 resulted in enhanced
   malignant behaviors. Investigations into the underlying mechanisms
   revealed that GJB5 is integral to the activation of the Akt-mTOR
   (mammalian target of rapamycin) signaling pathway. GJB5 knockdown or
   knockout led to diminished phosphorylation of Akt and S6 kinase,
   whereas GJB5 overexpression correlated with increased Akt-mTOR
   signaling in primary human cervical cancer cells. Additionally, we
   identified a novel interaction between GJB5 and the Gαi3 (G alpha
   inhibitory protein 3), underscoring the crucial role of GJB5 in
   mediating Akt activation via Gαi3. In vivo studies utilizing xenograft
   models provided further evidence for the oncogenic function of GJB5.
   The knockdown of GJB5 resulted in a marked reduction in the growth of
   cervical cancer xenografts. Observations of proliferation arrest,
   inactivation of the Akt-mTOR pathway, and the induction of apoptosis
   were noted in GJB5-depleted cervical cancer xenograft tissues.
   Collectively, these findings underscore GJB5 as a key oncogenic driver
   in cervical cancer and indicate that targeting GJB5 could be a
   promising therapeutic approach for this disease.

   Subject terms: Cervical cancer, Cervical cancer

Introduction

   Cervical cancer, a significant global health burden, primarily arises
   from persistent infection with high-risk human papillomavirus (HPV)
   genotypes [[30]1, [31]2]. This malignancy disproportionately affects
   women in low- and middle-income countries due to limited access to
   screening and vaccination programs [[32]3, [33]4]. There are an
   estimated 660,000 new cases and 350,000 deaths from cervical cancer
   globally, underscoring the need for effective prevention strategies
   [[34]5, [35]6]. Cervical cancer predominantly presents as squamous cell
   carcinoma (SCC) or adenocarcinoma, with the former accounting for about
   90% of cases, characterized by distinct cellular atypia and invasive
   growth patterns [[36]1]. Current treatment paradigms encompass surgical
   intervention, radiotherapy, and chemotherapy, with treatment strategies
   tailored to the stage of disease progression [[37]7]. However,
   challenges such as late-stage diagnosis, disparities in healthcare
   access, and suboptimal HPV vaccine uptake hinder effective management
   [[38]8]. Consequently, continued research aimed at improving early
   detection, refining therapeutic approaches, and enhancing outcomes for
   affected populations remains crucial [[39]1, [40]2].

   Current targeted therapies for cervical cancer primarily encompass
   monoclonal antibodies [[41]9–[42]12], such as bevacizumab [[43]13],
   which inhibits vascular endothelial growth factor (VEGF), and immune
   checkpoint inhibitors like pembrolizumab and nivolumab [[44]14], which
   enhance anti-tumor T-cell immunity by blocking programmed cell death
   protein 1 (PD-1) [[45]15, [46]16]. While these agents have demonstrated
   promising clinical efficacy in patients with recurrent or metastatic
   disease, resulting in improved progression-free and overall survival
   rates in select cancer populations, several limitations persist
   [[47]10, [48]11, [49]17]. These include inter-patient variability in
   treatment response, the potential for immune-related adverse events,
   and the emergence of treatment resistance over time [[50]10, [51]11,
   [52]17]. Furthermore, the current reliance on a limited number of
   therapeutic targets underscores the critical need to identify and
   validate novel molecular and genetic targets to expand the therapeutic
   armamentarium for cervical cancer [[53]9–[54]12]. This pursuit is
   essential for developing more personalized and effective treatment
   strategies that can improve outcomes for all patients. It is also the
   research focus of our group [[55]18].

   Gap junction protein beta-5 (GJB5), also known as connexin 31.1, is a
   member of the connexin family of proteins that form gap junctions,
   intercellular channels facilitating direct communication between
   adjacent cells [[56]19, [57]20]. It is primarily expressed in the inner
   ear, skin, and various epithelial tissues, playing an important role in
   maintaining cellular homeostasis and tissue integrity [[58]21–[59]24].
   GJB5 contributes to the formation of gap junction channels that allow
   the passage of ions and small molecules between adjacent cells, which
   is essential for processes such as cellular signaling and metabolic
   cooperation [[60]21–[61]25]. The mechanism of GJB5 involves the
   assembly of connexin proteins into hexameric structures, or connexons,
   that dock with those from neighboring cells to form functional gap
   junctions. GJB5 is defective in forming functional gap junctions on its
   own, but its interaction with co-expressed connexins like Cx43 can
   enhance its assembly into gap junctions and improve intercellular
   signaling [[62]25]. Studies have explored other functions of GJB5.
   Deficiency of GJB5 impaired the differentiation of trophoblast stem
   cells and hindered placental development [[63]21, [64]26]. GJB5
   deficiency in knockout mice increased exploratory behavior and impaired
   object recognition, indicating its role in learning and memory
   [[65]27].

   The intercellular communication processes by GJB5 are important for
   epidermal differentiation and the sensing of environmental stimuli
   [[66]21–[67]24]. GJB5’s link to epidermal differentiation is
   particularly relevant as cervical cancer frequently originates in the
   squamous epithelium, which shares characteristics with epidermal tissue
   [[68]1]. Furthermore, pan-cancer analyses indicate variable GJB5
   expression across malignancies, with notable reports of elevated GJB5
   mRNA expression in cervical squamous cell carcinoma [[69]28]. This
   distinct expression profile in cervical squamous cell carcinoma,
   alongside its fundamental role in modulating gap junction and
   intercellular communication [[70]21–[71]24], suggest a potential
   involvement in cervical carcinogenesis. This study aims to investigate
   the expression, functional role, and potential underlying mechanisms of
   GJB5 in cervical cancer, as these aspects remain largely unexplored.

Materials and methods

Reagents and antibodies

   Reagents and antibodies were obtained from the following sources:
   Puromycin, cell culture media, serum FBS, Matrigel matrix, polybrene,
   and CCK-8 were purchased from Sigma-Aldrich (St. Louis, MO). Terminal
   deoxynucleotidyl transferase dUTP nick end labeling (TUNEL),
   5-ethynyl-2’-deoxyuridine (EdU), 4’,6-diamidino-2-phenylindole (DAPI),
   and JC-1 were acquired from Thermo Fisher Scientific (Invitrogen Thermo
   Fisher Scientific, Carlsbad, CA). All primary antibodies utilized in
   this study were sourced from Cell Signaling Technology (Danvers, MA,
   USA) and Thermo Fisher Scientific (Invitrogen, Suzhou, China). The
   nucleotide sequences and PCR primers were synthesized by Genechem
   (Shanghai, China).

Cells

   As previously detailed [[72]18, [73]29], cervical cancer tissues and
   neighboring epithelial tissues were fragmented and digested with
   collagenase type I and dispase II (Sigma). The cells were washed,
   centrifuged, and incubated in a complete medium supplemented with
   penicillin/streptomycin and DNase (500 U). Following filtration and
   re-suspension, primary cervical cancer cells (“priCC-1”, “priCC-2”, and
   “priCC-3”, from three individuals) and cervical epithelial cells
   (“priCEpi-1” and “priCEpi-2”, from two individuals) were cultivated in
   the described medium [[74]29]. HeLa229 and Caski cervical cancer cell
   lines were obtained from the Cell Bank of the Institute of Biological
   Science at CAS (Shanghai, China). All procedures involving human cells
   were authorized by the Ethics Committee of The Affiliated Kunshan
   Hospital of Jiangsu University and conformed to the principles of the
   Helsinki Declaration.

Human tissues

   Cervical cancer tissues and corresponding paracancerous normal
   epithelial tissues were obtained from a cohort of twenty patients
   (n = 20) at the Affiliated Hospitals of Soochow University following
   the acquisition of written-informed consent from each participant.
   Pertinent patient-specific details have been previously reported
   [[75]29]. All of the cancers are identified as squamous cell carcinomas
   [[76]29]. Fresh tissue lysates were subjected to Western blotting
   analysis and quantitative real-time PCR (qPCR) for further
   investigation. All experimental procedures were granted approval by the
   Ethics Committee of the Affiliated Kunshan Hospital of Jiangsu
   University and were conducted in strict adherence to the ethical
   guidelines outlined in the Helsinki Declaration.

Immunohistochemistry (IHC)

   Immunohistochemistry (IHC) was conducted on paraffin-embedded tissue
   sections with a thickness of 4 μm. After standard de-paraffinization
   and rehydration, antigen retrieval was performed using citrate buffer.
   To inhibit endogenous peroxidase activity, the sections were treated
   with hydrogen peroxide. The primary antibody was then applied and
   incubated for 10 h. Following this, a secondary antibody and
   streptavidin-horseradish peroxidase conjugate were introduced, each
   incubated for 2 h. Finally, diaminobenzidine (DAB) was utilized to
   visualize the expression of the target antigen.

Western blotting and co-immunoprecipitation (Co-IP) assays

   In brief, protein extracts (30–40 µg) from cellular and tissue lysates
   were resolved using SDS-PAGE gels (10–15%) and transferred onto PVDF
   membranes. Following a blocking step, membranes were incubated with
   primary antibodies overnight at 4 °C, followed by incubation with
   designated secondary antibodies for 45 min at ambient temperature.
   Protein bands were visualized using enhanced chemiluminescence (ECL)
   and quantified using ImageJ software. The specific protocols for Co-IP
   have been detailed elsewhere [[77]30]. Uncropped blot images are
   presented in Fig. [78]S1.

qPCR

   In brief, RNA was isolated from cells and tissues using TRIzol
   (Biyuntian, Wuxi, China), reverse transcribed into complementary DNA
   (cDNA) with a Takara PCR amplification kit (Beijing, China), and
   quantified using qPCR with SYBR Green PCR Master Mixes (Beijing, China)
   on the ABI-7900 system. GAPDH was employed as the reference gene, and
   relative gene expression was determined using the 2^−ΔΔCt method.
   Primers were obtained from Genechem.

GJB5 gene silencing

   Two different short hairpin RNAs (shRNAs) targeting GJB5, designated
   kdGJB5-sh1 and kdGJB5-sh2, were synthesized and sub-cloned into the
   GV369 lentiviral vector obtained from Genechem (Shanghai, China).
   Lentiviral particles were generated by transfecting these constructs,
   along with requisite viral packaging plasmids, into HEK-293 cells.
   Subsequently, the resulting lentiviral particles were employed to
   transduce the described cervical cancer cells or epithelial cells.
   Following selection (with puromycin), stable cells exhibiting GJB5
   knockdown were established, with GJB5 expression validated through qPCR
   and Western blotting analysis.

GJB5 gene knockout

   The primary cervical cancer cells were cultured in complete growth
   medium supplemented with polybrene until they reached 60% confluence.
   Subsequently, they were transduced with a lentiviral vector expressing
   Cas9 nuclease, kindly provided by Dr. Cao [[79]29], to generate stable
   cells. These cells were then subjected to transduction with two
   distinct CRISPR/Cas9 constructs, each engineered to mediate GJB5 gene
   knockout and harboring unique single-guide RNA (sgRNA) sequences:
   koGJB5-sg1 and koGJB5-sg2. Following puromycin selection to enrich for
   transduced cells, single-cell clones were isolated by plating the cells
   in 96-well plates. The successful disruption of GJB5 gene expression
   was subsequently validated through DNA sequencing and Western blotting
   analysis. Control cells were generated by transducing cells with a
   lentiviral vector expressing Cas9 nuclease and a non-targeting
   scrambled control sgRNA (“koC”), also obtained from Dr. Cao [[80]29].

GJB5 overexpression

   GJB5 overexpression was achieved using the hGJB5-expressing lentiviral
   GV369 construct, sourced from Genechem. The production of lentivirus
   involved transfecting the construct with packaging plasmids into
   HEK-293 cells. Following this, the resulting viral particles were used
   to infect cultured cervical cancer cells, which transitioned to
   puromycin-containing medium after 48 h. Stable cells with GJB5
   overexpression were established after five passages, with GJB5
   overexpression validation performed using qPCR and Western blotting
   techniques.

Gαi3 (G alpha inhibitory protein 3) silencing or overexpression

   Silencing of Gαi3 was achieved using a lentiviral shRNA (shGαi3), while
   overexpression was accomplished with a lentiviral construct (oeGαi3),
   both provided by Dr. Cao [[81]29, [82]31, [83]32]. The stable cell
   selection procedure for these manipulations has been previously
   described [[84]29, [85]31, [86]32].

CCK-8 assay

   Cell viability was evaluated utilizing the CCK-8 method. In detail,
   3000 cells per well were placed into a 96-well plate and maintained for
   96 h. Following this, 20 μL of CCK-8 solution was introduced to each
   well and incubated for 80 min. Viability was determined by measuring
   the absorbance at 440 nm with a microplate reader, indicating the
   reduction of the CCK-8 reagent.

Colony formation assay

   In brief, 13,000 cervical cancer cells per dish, subjected to specific
   genetic modifications, were seeded in 10-cm culture dishes. After a
   10-day incubation period, colonies were stained and counted manually.

Nuclear EdU/TUNEL staining

   Cervical cancer or epithelial cells were cultured in 12-well plates for
   predetermined times. They were fixed using paraformaldehyde and
   permeabilized with Triton X-100. Staining was performed using TUNEL/EdU
   and DAPI dyes. Stained cells were imaged with a Zeiss fluorescence
   microscope (Oberkochen, Germany). The ratio of EdU/TUNEL-positive cells
   to DAPI-stained nuclei was assessed.

Transwell assay

   The specific cervical cancer cells, at a density of 8000 cells per
   condition, were placed on the upper side of Transwell inserts to
   migrate over 24 h. Cells that traversed to the lower surface were fixed
   and stained with crystal violet. For in vitro cell invasion assays,
   Matrigel was coated to the inserts.

JC-1 staining

   The mitochondrial membrane potential (MMP) was evaluated using a JC-1
   assay kit from Invitrogen, following the manufacturer’s guidelines. The
   cervical cancer cells subjected to genetic modifications were incubated
   with JC-1 dye (3.5 μg/mL) for 25 min, and excess dye was removed
   through washing with warm PBS. The resulting green fluorescence,
   indicating reduced MMP, was measured at an excitation wavelength of
   480 nm using fluorescence microscopy.

Cytosolic Cytochrome c detection

   Levels of Cytochrome c were measured via an enzyme-linked immunosorbent
   assay (ELISA) provided by Thermo-Fisher Invitrogen. This assay involves
   immobilizing specific capture antibodies against Cytochrome c on a
   microplate. Cytosolic protein lysates (0.8 μg/μL, 24 μg per sample)
   were added, allowing Cytochrome c to bind to the capture antibodies. A
   detection antibody linked to an enzyme was then introduced, forming a
   complex with the captured Cytochrome c. A substrate solution was added,
   resulting in an enzyme-driven colorimetric reaction, detected at
   450 nm. A standard curve was created to determine Cytochrome c levels
   based on absorbance values obtained.

Caspase-3 and Caspase-9 activity assessment

   The activities of Caspase-3 and Caspase-9 were evaluated utilizing
   colorimetric assay kits (Thermo-Fisher Invitrogen), following the
   protocols provided by the manufacturer. In summary, cell or tissue
   lysates were prepared at a concentration of 0.8 μg/μL, with 24 μg
   allocated for each sample. These lysates were then incubated with
   specific substrates designed for caspases. The enzymatic cleavage of
   these substrates resulted in the formation of a colorimetric product,
   which was subsequently measured spectrophotometrically at a wavelength
   of 400 nm. A standard curve was consistently generated to accurately
   determine caspase activity.

Constitutively-active mutant Akt1 (caAkt1)

   Specifically, the lentivirus expressing the caAkt1 (S473D) variant, as
   previously documented (provided by Dr. Cao [[87]29, [88]31, [89]32]),
   was introduced to cultured primary human cervical cancer cells.
   Following this introduction, stable cells expressing caAkt1 were
   established through selection with puromycin, with its expression
   validated through Western blotting assays.

Xenograft assays

   Female nude BALB/c mice, weighing between 17.7 and 18.2 grams, were
   obtained from Shanghai SLAC Laboratory Animal Co. (Shanghai, China). A
   total of seven million genetically modified primary cervical cancer
   cells were subcutaneously injected into the flanks of the mice. Tumor
   volumes, body weights, and daily growth rates (measured in mm³ per day)
   were recorded 18 days after cell inoculation in accordance with
   established guidelines [[90]33]. TUNEL staining was subsequently
   performed on tumor sections using a Biyuntian kit (Wuxi, China),
   followed by washing and counterstaining with DAPI to visualize cell
   nuclei. All animal procedures were approved by the Institutional Animal
   Care and Use Committee (IACUC) and the Ethics Review Board of The
   Affiliated Kunshan Hospital of Jiangsu University.

Statistical analysis

   All in vitro experiments were conducted with five biological
   replicates. Data exhibiting normal distribution are presented as
   mean ± standard deviation (SD). Unpaired Student’s t-tests were
   employed to compare data between two experimental groups. For
   comparisons among three or more groups, one-way analysis of variance
   (ANOVA) was conducted, followed by post-hoc multiple comparison tests
   (Scheffé's and Tukey’s method). Statistical significance was considered
   at a P-value threshold of less than 0.05.

Results

GJB5 expression and clinical outcomes in cervical cancer

   Gene expression analysis of cervical cancer specimens from The Cancer
   Genome Atlas (TCGA) revealed significant dysregulation of GJB5
   expression. Compared to normal cervical tissue (“Normal”), GJB5
   expression was markedly elevated in adenocarcinoma, squamous cell
   carcinoma (SCC), and adenosquamous carcinoma (Fig. [91]1A). This
   upregulation was further corroborated by paired sample analysis, which
   demonstrated a significant increase in GJB5 expression in squamous cell
   carcinoma compared to normal tissues from the same patients (Fig.
   [92]1B). With respect to histological grade, G2 tumors displayed the
   highest levels of GJB5 expression (Fig. [93]1C). To investigate the
   clinical significance of GJB5 expression, Kaplan-Meier survival
   analysis was conducted. Results demonstrated a significant association
   between high GJB5 overexpression and reduced overall survival in
   cervical cancer patients (Fig. [94]1D). This adverse prognostic impact
   of elevated GJB5 expression was particularly evident in patients with
   squamous cell carcinoma (Fig. [95]1E). In contrast, no significant
   correlation was observed between GJB5 expression and overall survival
   in adenocarcinoma patients (Fig. [96]1F).

Fig. 1. GJB5 expression and clinical outcomes in cervical cancer.

   [97]Fig. 1
   [98]Open in a new tab

   Box plot showing GJB5 mRNA expression levels in normal tissues,
   adenocarcinoma, squamous cell carcinoma, and adenosquamous carcinoma
   tissues (A). Box plot comparing GJB5 mRNA expression in paired normal
   and squamous cell carcinoma tissue samples (B). Box plot showing GJB5
   expression levels in G1, G2, and G3&G4 cervical cancer tumors (C).
   Kaplan–Meier survival curves for patients with high and low GJB5
   expression in cervical cancer patients (D), squamous cell carcinoma
   patients (E) and adenocarcinoma patients (F). Receiver Operating
   Characteristic (ROC) curve for GJB5 mRNA expression in differentiating
   cervical cancer from normal tissue samples (G). ROC curve for GJB5 mRNA
   expression in differentiating squamous cell carcinoma from
   adenocarcinoma (H). Error bars represent standard deviation. “TPM”
   stands for transcripts per million. “AUC” stands for area under curve.
   “CI” stands for confidence interval. “HR” stands for hazard rate. “TPR”
   stands for true positive rate. “FPR” stands for false positive rate.
   Data are presented as mean ± standard deviation (SD). *P < 0.05.
   **P < 0. 01. ***P < 0.001.

   We also studied other GJB family proteins whose expression/roles in
   cervical cancer had not been previously reported, including GJB3 and
   GJB4. Although analysis of TCGA data indicates upregulated expression
   of GJB3 and GJB4 mRNA in cervical cancer (Fig. [99]S2A, B), their
   overexpression is not significantly associated with poorer patient
   prognosis (Fig. [100]S2C, D).

   To assess the diagnostic potential of GJB5 expression, receiver
   operating characteristic (ROC) curve analysis was performed. Figure
   [101]1G demonstrated excellent discriminatory power for GJB5 in
   distinguishing cervical cancer tissues from normal tissues, with an
   area under the curve (AUC) of 0.973. Furthermore, Fig. [102]1H revealed
   that GJB5 expression can effectively differentiate between squamous
   cell carcinoma and adenocarcinoma, with an AUC of 0.911. These findings
   collectively suggest that GJB5 expression may serve as a valuable
   biomarker for the diagnosis and subtyping of cervical cancer.

The single-cell RNA sequencing demonstrates overexpression of GJB5 in the
epithelial population of cervical squamous cell carcinoma

   To gain a deeper understanding of the cellular heterogeneity and gene
   expression dynamics in cervical cancer, we evaluated the cervical
   squamous carcinoma single-cell RNA sequencing data from the
   ArrayExpress database with the accession number E-MTAB-11948. This
   approach allows for the identification of cell-type-specific gene
   expression patterns. The results revealed a distinct expression pattern
   of GJB5 in cervical squamous cell carcinoma. UMAP (uniform manifold
   approximation and projection) dimensionality reduction of the dataset
   identified distinct cell clusters corresponding to various cell types,
   including epithelial, lymphocyte, fibroblast, endothelial, muscle,
   macrophage, and neutrophil cells (Fig. [103]2A). The expression of GJB5
   was predominantly localized to epithelial cells, as shown by the
   density plot (Fig. [104]2B, C). Further analysis revealed a significant
   increase in GJB5 expression in epithelial cells from cervical squamous
   cell carcinoma samples compared to paracancer normal tissue (Fig.
   [105]2B, C).

Fig. 2. The single-cell RNA sequencing demonstrates overexpression of GJB5 in
the epithelial population of cervical squamous cell carcinoma.

   [106]Fig. 2
   [107]Open in a new tab

   UMAP dimensionality reduction of single-cell RNA-seq data from cervical
   squamous cell carcinoma samples, showing distinct cell clusters (A).
   Density plot showing the expression of GJB5 mRNA across different cell
   types, demonstrating its enrichment in epithelial cells (B). Comparison
   of GJB5 expression in epithelial cells from cervical squamous cell
   carcinoma and paracancer normal tissues (C). Volcano plot showing the
   correlation between GJB5 expression and other genes within the
   epithelial cell cluster (D). Genes with a correlation coefficient >0.3
   and P-value < 0.05 are highlighted (D). Gene ontology (GO) enrichment
   analysis of genes highly correlated with GJB5, showing enrichment in
   biological processes related to cell motility and morphology (E).
   Reactome pathway enrichment analysis of genes highly correlated with
   GJB5, revealing significant enrichment in Rho GTPase signaling pathways
   (F).

   To explore the potential functional implications of GJB5 upregulation
   in cervical squamous cell carcinoma, we performed correlation analysis
   of GJB5 expression with all other genes within the epithelial cell
   cluster. Genes exhibiting a correlation coefficient greater than 0.3
   and a P-value less than 0.05 were considered highly correlated with
   GJB5 (Fig. [108]2D). Gene ontology (GO) enrichment analysis of these
   genes revealed significant enrichment in biological processes related
   to lamellipodium assembly, regulation of stress fiber assembly, and
   morphogenesis of a polarized epithelium (Fig. [109]2E). Additionally,
   pathway enrichment analysis using Reactome identified significant
   enrichment in pathways involving Rho GTPases, which regulate actin
   cytoskeleton dynamics (Fig. [110]2F). Thus, the scRNA-seq analysis
   demonstrates overexpression of GJB5 in the epithelial population of
   cervical squamous cell carcinoma, suggesting its potential involvement
   in tumor progression and highlighting its potential as a therapeutic
   target.

GJB5 overexpression in cervical cancer tissues from locally treated patients
and different cervical cancer cells

   Next, our study revealed a significant upregulation of GJB5 in cervical
   cancer tissues of locally-treated patients. Analysis of cervical cancer
   tissues from twenty (n = 20) patients (as reported previously
   [[111]29]) indicated a substantial increase in GJB5 mRNA levels when
   compared to matched paracancerous tissues (Fig. [112]3A). This elevated
   mRNA expression was further confirmed at the protein level. Western
   blotting analysis conducted on four representative patients (Patient-1#
   to Patient-4#) demonstrated markedly heightened GJB5 protein expression
   in cervical cancer tissues relative to their paracancerous normal
   counterparts (Fig. [113]3B). Comprehensive analysis of all 20 patient
   samples reinforced the significant upregulation of GJB5 protein in
   cervical cancer tissues (Fig. [114]3C).

Fig. 3. GJB5 overexpression in cervical cancer tissues from locally-treated
patients and different cervical cancer cells.

   [115]Fig. 3
   [116]Open in a new tab

   The expression levels of the listed genes and proteins in cervical
   cancer tissues (“T”) and matched normal paracancerous cervical
   epithelial tissues (“N”) were evaluated in a cohort of twenty (n = 20)
   primary cervical cancer patients, with results quantified (A–C).
   Additionally, GJB5 mRNA and protein expression were assessed in the
   specified cervical cancer cells and cervical epithelial cells (D, E).
   Data are presented as mean ± standard deviation (SD) with n = 5
   replicates. Asterisks (*) indicate statistically significant
   differences (P < 0.05) compared to the “N” tissues or priCEpi-1 cells.
   The experiments were independently replicated five times with
   consistent results.

   To further investigate GJB5 expression in cervical cancer cells, we
   examined both primary and immortalized cells. Our results indicated
   that both GJB5 mRNA (Fig. [117]3D) and protein (Fig. [118]3E) levels
   were significantly elevated in primary human cervical cancer cells
   (priCC-1 and priCC-2) and immortalized cervical cancer cell lines
   (Caski and HeLa229) compared to primary cervical epithelial cells
   (priCEpi-1 and priCEpi-2). Collectively, these findings underscore a
   consistent and significant overexpression of GJB5 in cervical cancer
   tissues and cells, suggesting its potential role in the pathogenesis of
   this disease.

GJB5 silencing inhibits malignant behaviors in cervical cancer cells

   To investigate the possible functional role of GJB5 in cervical cancer
   malignant behaviors, we utilized lentiviral shRNA to knockdown (kd)
   GJB5 expression in priCC-1 primary human cervical cancer cells. Two
   distinct shRNA sequences (kdGJB5-sh1 and kdGJB5-sh2) were employed to
   achieve robust silencing. qPCR analysis revealed a significant decrease
   in GJB5 mRNA expression in priCC-1 cells transfected with both shRNA
   constructs compared to control cells with scramble control shRNA
   (“kdC”) (Fig. [119]4A). Western blotting analysis also confirmed a
   significant reduction in GJB5 protein levels (Fig. [120]4B). The GJB4
   and GJB5 genes encode closely related beta-connexin proteins that play
   roles in forming gap junctions and facilitating direct intercellular
   communication [[121]34].The silencing of GJB5 did not affect the
   expression levels of GJB4 (Fig. [122]4A, B). Functional assays
   demonstrated a pronounced inhibitory effect of GJB5 silencing on key
   aspects of cervical cancer cell behaviors. Colony formation assays
   showed a significant reduction in the number of colonies formed by
   GJB5-silenced priCC-1 cells compared to control cells (Fig. [123]4C),
   indicating impaired growth potential. Furthermore, the nuclear EdU
   incorporation assays revealed a decrease in cell proliferation in
   GJB5-deficient cells (Fig. [124]4D). Consistent with these findings,
   CCK-8 assays demonstrated a significant reduction in cell viability in
   GJB5-silenced cells (Fig. [125]4E). To assess the impact of GJB5 on
   cell motility, we performed cell migration (“Transwell”) and invasion
   (“Matrigel Transwell”) assays. In both assays, GJB5-silenced priCC-1
   cells exhibited significantly reduced migration and invasion
   capabilities compared to control cells (Fig. [126]4F, G). These
   findings collectively suggest that GJB5 plays a crucial role in
   promoting the growth, proliferation, survival and motility of cervical
   cancer cells.

Fig. 4. GJB5 silencing inhibits malignant behaviors in cervical cancer cells.

   [127]Fig. 4
   [128]Open in a new tab

   qPCR (A) and western blotting (B) analysis showing GJB5/GJB4 mRNA and
   protein expression levels in priCC-1 primary human cervical cancer
   cells transfected with control shRNA (“kdC”) or GJB5-specific shRNA
   (kdGJB5-sh1 and kdGJB5-sh2). Cells were subsequently cultured in
   complete medium for designated time periods to assess colony formation
   (C), cell proliferation (by measuring the ratio of EdU-positive nuclei,
   D) and cell viability (via CCK-8 OD, E), as well as in vitro cell
   migration (using “Transwell” assays, F) and invasion (through “Matrigel
   Transwell” assays, G). Additionally, other patient-derived primary
   human cervical cancer cells (priCC-2) (H–L), along with the established
   Caski and HeLa229 cell lines (H–L), and primary human cervical
   epithelial cells (priCEpi-1 and priCEpi-2, derived from two patients)
   (M–O), were transduced with kdGJB5-sh2 or kdC. Stable cells were
   established following puromycin selection. The expression levels of
   specific mRNAs were evaluated (H, I, M). Cells were again cultured in
   complete medium for the specified time periods, and measurements for
   cell proliferation (J, N), cell viability (CCK-8 OD, K, O), and in
   vitro cell migration (L) were performed. “Pare” stands for parental
   control cells. Data are presented as mean ± standard deviation (SD)
   with n = 5 replicates. Asterisks (*) indicate statistically significant
   differences (P < 0.05) compared to the “kdC” treatment. “N.S.” denotes
   no statistically significant difference (P > 0.05). The experiments
   were independently replicated five times with consistent results. Scale
   bar represents 100 μm.

   To validate the role of GJB5 in multiple cervical cancer contexts, we
   expanded our investigation to additional cells, including the primary
   priCC-2 cervical cancer cells and the immortalized Caski and HeLa229
   cell lines. Using the same kdGJB5-sh2 lentiviral construct, we observed
   consistent and robust downregulation of GJB5 mRNA expression across all
   three cervical cancer cell types (Fig. [129]4H), while GJB4 mRNA levels
   remained unaffected (Fig. [130]4I). This selective silencing of GJB5
   led to a significant decrease in cell proliferation, as measured by
   nuclear EdU incorporation (Fig. [131]4J), and a reduction in cell
   viability, as assessed by CCK-8 assays (Fig. [132]4K). Furthermore,
   cell migration assays demonstrated a significant decrease in migratory
   capacity in GJB5-silenced cells compared to controls (Fig. [133]4L). To
   examine the impact of GJB5 on normal cervical epithelial cells, we
   utilized primary human cervical epithelial cells, priCEpi-1 and
   priCEpi-2. Similar to the cancer cells, GJB5 mRNA expression was
   significantly downregulated in these cells following transfection with
   the kdGJB5-sh2 construct (Fig. [134]4M). However, in contrast to the
   cervical cancer cells, GJB5 silencing in primary epithelial cells did
   not result in a significant inhibition of cell proliferation (Fig.
   [135]4N) or a decrease in cell viability (Fig. [136]4O). The reason for
   the lack of effect in primary epithelial cells could be due to the
   relatively low expression of GJB5 in normal versus cancerous cells (see
   Figs. [137]1–[138]3). Thus, these findings demonstrate that GJB5 plays
   an important role in promoting the survival, growth and proliferation
   of cervical cancer cells, while having limited impact on normal
   cervical epithelial cells.

GJB5 silencing results in apoptosis activation in cervical cancer cells

   To investigate the potential role of GJB5 on apoptosis, we again
   employed lentiviral shRNA to knockdown GJB5 expression in priCC-1
   primary cervical cancer cells. Two distinct shRNA sequences (kdGJB5-sh1
   and kdGJB5-sh2) were utilized to ensure the specificity of the observed
   effects (see Fig. [139]4). Our findings demonstrate that silencing GJB5
   significantly induced apoptosis. We observed a significant increase in
   the activities of both Caspase-3 (Fig. [140]5A) and Caspase-9 (Fig.
   [141]5B), key executioners of apoptosis, in GJB5-silenced priCC-1
   cells. This was further corroborated by the increased cleavage of
   Caspase-3 and its downstream target, PARP1 [Poly(ADP-ribose) polymerase
   1], as detected by Western blotting analysis (Fig. [142]5C). Moreover,
   the levels of cytosol cytochrome C were significantly increased in GJB5
   shRNA-expressing cells (Fig. [143]5D). In addition, JC-1 staining
   revealed a significant shift towards the green monomer form of JC-1,
   indicating a loss of mitochondrial membrane potential (MMP) (Fig.
   [144]5E). This depolarization is a hallmark of the intrinsic apoptotic
   pathway. The nuclear TUNEL staining demonstrated a significant increase
   in the number of cells with fragmented DNA, indicating apoptosis (Fig.
   [145]5F).

Fig. 5. GJB5 silencing results in apoptosis activation in cervical cancer
cells.

   [146]Fig. 5
   [147]Open in a new tab

   The priCC-1 primary human cervical cancer cells with control shRNA
   (“kdC”) or GJB5-specific shRNA (kdGJB5-sh1 and kdGJB5-sh2) were
   cultured in complete medium for 72 h, and were subjected to Caspase-3
   activity assay (A), Caspase-9 activity assay (B), Western blotting
   analysis of cleaved-Caspase-3 and cleaved-PARP1 (C), cytosol Cytochrome
   C release (D) and JC-1 staining for mitochondrial membrane potential
   (E). The nuclear TUNEL staining was carried out to detect DNA
   fragmentation (F). Additionally, other patient-derived primary human
   cervical cancer cells (priCC-2) (G, H), the established Caski and
   HeLa229 cell lines (G, H), and along with primary human cervical
   epithelial cells (priCEpi-1 and priCEpi-2, derived from two patients)
   (I) were transduced with kdGJB5-sh2 or kdC. Stable cells were
   established following puromycin selection. Cells were again cultured in
   complete medium for 72 h, and measurements include JC-1 staining for
   mitochondrial membrane potential (G) and TUNEL staining to detect DNA
   fragmentation (H, I). “Pare” stands for parental control cells. Data
   are presented as mean ± standard deviation (SD) with n = 5 replicates.
   Asterisks (*) indicate statistically significant differences (P < 0.05)
   compared to the “kdC” treatment. “N.S.” denotes no statistically
   significant difference (P > 0.05). The experiments were independently
   replicated five times with consistent results. Scale bar represents 100
   μm.

   To further investigate the role of GJB5 in apoptosis, we extended our
   studies to additional cervical cancer cells, including primary priCC-2
   cells and the immortalized Caski and HeLa229 lines. Using the same
   kdGJB5-sh2 lentiviral construct (see Fig. [148]4), we observed
   consistent results in these cancer cell types: silencing GJB5
   expression resulted in mitochondrial depolarization, as indicated by a
   significant increase in the green monomer form of JC-1 (Fig. [149]5G),
   and increased apoptosis, as confirmed by a significant increase in the
   number of TUNEL-positive cells (Fig. [150]5H). In contrast, silencing
   GJB5 expression in primary human cervical epithelial cells (priCEpi-1
   and priCEpi-2) using the same kdGJB5-sh2 construct (see Fig. [151]4)
   did not significantly induce apoptosis, as the TUNEL-positive nuclei
   ratio was not significantly changed (Fig. [152]5I). These findings
   suggest that the pro-apoptotic effect of GJB5 silencing is specific to
   cervical cancer cells.

GJB5 knockout inhibits malignant behaviors in cervical cancer cells

   To confirm the specificity of the observed effects of GJB5 silencing,
   we employed CRISPR/Cas9-mediated gene editing to generate GJB5 knockout
   (koGJB5) in priCC-1 cervical cancer cells. Two distinct single guide
   RNA (sgRNA) sequences (koGJB5-sg1 and koGJB5-sg2) were utilized to
   target GJB5, resulting in robust depletion of GJB5 protein without
   affecting the expression of GJB4 (Fig. [153]6A). GJB5 KO significantly
   impacted malignant behaviors in priCC-1 cells. Colony formation assays
   demonstrated a significant decrease in the number of colonies formed by
   koGJB5 cells compared to control cells (koC), indicating reduced cell
   proliferation (Fig. [154]6B). Cell viability, tested via CCK-8 assay,
   was also decreased (Fig. [155]6C). This was further supported by EdU
   incorporation assays, which showed a significant reduction in DNA
   synthesis in koGJB5 cells (Fig. [156]6D). Additionally, “Transwell”
   assays revealed a significant reduction in cell migration capacity in
   koGJB5 cells (Fig. [157]6E). koGJB5 cells also exhibited a significant
   increase in the green monomer form of JC-1, indicating mitochondrial
   depolarization (Fig. [158]6F), and increased apoptosis, as confirmed by
   a significant increase in the number of TUNEL-positive cells (Fig.
   [159]6G).

Fig. 6. GJB5 knockout inhibits malignant behaviors in cervical cancer cells.

   [160]Fig. 6
   [161]Open in a new tab

   The priCC-1 (A–H) or pri-CC-2 (I–L) primary human cervical cancer cells
   transfected with the lentiviral CRIPR/Cas9 construct containing control
   sgRNA (“koC”) or koGJB5-sg1/koGJB5-sg2 were established, the expression
   levels of listed proteins served were tested (A, H). Cells were
   subsequently cultured in complete medium for designated time periods to
   assess colony formation (B), cell viability (CCK-8 OD, C), cell
   proliferation (by measuring the ratio of EdU-positive nuclei, D and I),
   and in vitro cell migration (using “Transwell” assays, E, J). JC-1
   staining for mitochondrial membrane potential (F) and nuclear TUNEL
   staining to detect cell apoptosis (G, K) were also carried out. Data
   are presented as mean ± standard deviation (SD) with n = 5 replicates.
   Asterisks (*) indicate statistically significant differences (P < 0.05)
   compared to the “koC” treatment. “N.S.” denotes no statistically
   significant difference (P > 0.05). The experiments were independently
   replicated five times with consistent results. Scale bar represents 100
   μm.

   CRISPR/Cas9-mediated gene editing was extended to priCC-2 primary
   cervical cancer cells using the koGJB5-sg1 construct. Western blotting
   analysis confirmed efficient depletion of GJB5 protein in priCC-2 cells
   transfected with koGJB5-sg1 (Fig. [162]6H). Consistent with the
   findings in priCC-1 cells, GJB5 knockout in priCC-2 cells led to a
   significant decrease in cell proliferation, as evidenced by reduced EdU
   incorporation (Fig. [163]6I). Furthermore, “Transwell” assay results
   revealed a significant impairment in cell migration capacity in
   koGJB5-sg1-transfected priCC-2 cells (Fig. [164]6J). TUNEL staining
   demonstrated a significant increase in the number of cells with
   positive TUNEL staining after koGJB5-sg1 treatment, indicating
   apoptosis activation (Fig. [165]6K). These findings, in conjunction
   with the shRNA-mediated knockdown experiments, strongly suggest that
   GJB5 plays a critical role in regulating malignant cellular functions
   in cervical cancer cells.

GJB5 overexpression promotes malignant behaviors in cervical cancer cells

   Next, we propose that ectopic GJB5 overexpression might promote
   malignant behaviors of cervical cancer cells. Therefore, priCC-1
   primary cells were transduced with a lentiviral vector expressing GJB5.
   Two stable cell selections with increased GJB5 expression, designated
   oeGJB5-sL1 and oeGJB5-sL2, were established. qPCR analysis confirmed a
   significant increase in GJB5 mRNA levels in both oeGJB5-sL1 and
   oeGJB5-sL2 cells compared to the vector control (Vec) (Fig. [166]7A).
   Western blotting analysis further validated these findings, showing
   increased GJB5 protein expression in these cells (Fig. [167]7B). GJB5
   overexpression did not significantly alter the expression levels of
   GJB4 in priCC-1 cells (Fig. [168]7A, B). GJB5 overexpression
   significantly promoted cell proliferation (nuclear EdU incorporation)
   in oeGJB5-sL1 and oeGJB5-sL2 cells (Fig. [169]7C). Moreover, CCK-8 cell
   viability was increased in GJB5-overexpressing cells (Fig. [170]7D).
   Further assays revealed a significant increase in cell migration and
   invasion capacities in oeGJB5-sL1 and oeGJB5-sL2 cells (Fig. [171]7E,
   F). Next, we transduced primary priCC-2 cells and the immortalized
   Caski and HeLa229 cell lines with the same lentiviral vector expressing
   GJB5. Stable cells, oeGJB5, were established. qPCR analysis confirmed a
   significant increase in GJB5 mRNA levels in all three cell types
   compared to the vector control (Fig. [172]7G), leaving GJB4 mRNA
   expression unchanged (Fig. [173]7H). EdU incorporation assays
   demonstrated a significant increase in DNA synthesis in oeGJB5 cells
   compared to the vector control in all three cell types (Fig. [174]7I).
   Moreover, Transwell migration assays revealed a significant increase in
   cell migration capacity in oeGJB5 cervical cancer cells (Fig. [175]7J).
   Thus, GJB5 overexpression promotes several key malignant behaviors in
   cervical cancer cells, including increased proliferation, migration,
   and invasion.

Fig. 7. GJB5 overexpression promotes malignant behaviors in cervical cancer
cells.

   [176]Fig. 7
   [177]Open in a new tab

   GJB5 and GJB4 mRNA expression levels in priCC-1 cells transduced with
   the control vector (“Vec”) or the lentiviral GJB5-expressing construct
   (oeGJB5-sL1 or oeGJB5-sL2, two stable cell selections) were tested via
   quantitative PCR analysis (A), with Western blotting analysis of GJB5
   and GJB4 protein expression in these priCC-1 cells (B). Cells were
   subsequently cultured in complete medium for designated time periods to
   assess cell proliferation (by measuring the ratio of EdU-positive
   nuclei, C), cell viability (via CCK-8 OD, D), and in vitro cell
   migration (using “Transwell” assays, E) and invasion (through “Matrigel
   Transwell” assays, F). Additionally, other patient-derived primary
   human cervical cancer cells (priCC-2) along with the established Caski
   and HeLa229 cell lines were stably transduced with the lentiviral
   GJB5-expressing construct (oeGJB5) or control vector (“Vec”), GJB5 and
   GJB4 mRNA expression levels were tested (G and H); Cells were
   subsequently cultured in complete medium for designated time periods to
   assess cell proliferation (by measuring the ratio of EdU-positive
   nuclei, I) and migration (using “Transwell” assays, J). Data are
   presented as mean ± standard deviation (SD) with n = 5 replicates.
   Asterisks (*) indicate statistically significant differences (P < 0.05)
   compared to the “Vec” treatment. “N.S.” denotes no statistically
   significant difference (P > 0.05). The experiments were independently
   replicated five times with consistent results. Scale bar represents 100
   μm.

GJB5 positively regulates the Akt-mTOR signaling pathway in cervical cancer
cells

   The observation that GJB5 silencing or depletion exerts pleiotropic
   effects on cervical cancer cell behaviors, including inhibition of
   viability, proliferation, and migration, and enhancement of apoptosis
   (see Figs. [178]4–[179]6), suggests that GJB5 may regulate key
   oncogenic signaling cascades [[180]35–[181]38]. Recognizing the
   Akt-mTOR pathway as a key driver of cervical cancer malignancy
   [[182]37, [183]38], we focused our investigation on this pathway. In
   priCC-1 primary human cervical cancer cells, GJB5 silencing via shRNA
   (kdGJB5-sh1, kdGJB5-sh2, see Figs. [184]4 and [185]5) or
   CRISPR/Cas9-mediated knockout (koGJB5-sg1, koGJB5-sg2, see Fig. [186]6)
   significantly reduced phosphorylation of Akt (Ser-473) and its
   downstream target, S6K1, as determined by western blotting analysis
   (Fig. [187]8A, B). Conversely, GJB5 overexpression (oeGJB5-sL1,
   oeGJB5-sL2, see Fig. [188]7) augmented Akt-S6K1 phosphorylation (Fig.
   [189]8C). Expression levels of total Akt1 and S6K1 were unchanged
   following GJB5 depletion or overexpression in priCC-1 cells (Fig.
   [190]8A–C). These findings suggest that GJB5 positively regulates the
   Akt-mTOR signaling pathway in cervical cancer cells. To determine
   whether Akt inactivation mediates the anti-cervical cancer effects of
   GJB5 silencing, we introduced a constitutively-active Akt1 (caAkt1,
   S473D) mutant into GJB5-silenced priCC-1 cells, and it completely
   restored Akt-S6K1 phosphorylation without affecting GJB5 protein
   expression (Fig. [191]8D). Introduction of caAkt1 ameliorated the
   inhibitory effects of GJB5 silencing on priCC-1 cell proliferation
   (Fig. [192]8E) and migration (Fig. [193]8F). In addition,
   kdGJB5-sh2-induced mitochondrial depolarization (JC-1 green monomer
   accumulation, Fig. [194]8G) and apoptosis (Fig. [195]8H) were also
   inhibited with caAkt1 treatment in priCC-1 cells. These findings
   suggest that GJB5 plays an important role in promoting the Akt-mTOR
   signaling pathway in cervical cancer cells.

Fig. 8. GJB5 positively regulates the Akt-mTOR signaling pathway in cervical
cancer cells.

   [196]Fig. 8
   [197]Open in a new tab

   Expression levels of the listed proteins in the priCC-1 primary human
   cervical cancer cells with the control shRNA (“kdC”), the GJB5-specific
   shRNA (kdGJB5-sh1, kdGJB5-sh2), the lentiviral CRIPR/Cas9 construct
   containing control sgRNA (“koC”), the lentiviral CRIPR/Cas9 construct
   expressing koGJB5-sg1 or koGJB5-sg2, the control vector (“Vec”) or the
   lentiviral GJB5-expressing construct (oeGJB5-sL1 or oeGJB5-sL2, two
   stable cell selections) were shown (A–C). The kdGJB5-sh2-expressing
   priCC-1 primary cancer cells were further transduced with or without a
   lentiviral constitutively-active Akt1 (caAkt1, S473D) mutant, with
   stable cells formed. Expression of listed proteins was shown (D). Cells
   were subsequently cultured in complete medium for designated time
   periods to assess cell proliferation (by measuring the ratio of
   EdU-positive nuclei, E) and in vitro cell migration (using “Transwell”
   assays, F); JC-1 staining for mitochondrial membrane potential (G) and
   nuclear TUNEL staining to detect cell apoptosis (H) were also carried
   out. Data are presented as mean ± standard deviation (SD) with n = 5
   replicates. Asterisks (*) indicate statistically significant
   differences (P < 0.05) compared to the “kdC”/“koC”/“Vec” treatment. ^#
   indicates P < 0.05 (D–H). “N.S.” denotes no statistically significant
   difference (P > 0.05). The experiments were independently replicated
   five times with consistent results. Scale bar represents 100 μm.

GJB5 interacts with Gαi3 to promote Akt signaling activation in cervical
cancer cells

   Results in Fig. [198]8 confirmed that GJB5 is important for Akt-mTOR
   cascade activation, we tested its possible underlying mechanism. A
   recent study has shown that Gαi3 protein is essential in regulating
   Akt-mTOR cascade activation in cervical cancer [[199]29], possibly by
   associating with multiple receptor tyrosine kinases (RTKs) and non-RTK
   receptors [[200]31, [201]32, [202]39–[203]42]. We therefore explored
   GJB5’s potential interaction with Gαi3. Co-IP assays in priCC-1 and
   priCC-2 primary cervical cancer cells revealed a physical interaction
   between GJB5 and Gαi3 (Fig. [204]9A). This interaction was further
   validated in human cervical cancer tissues of three representative
   patients mentioned in Fig. [205]3 (Fig. [206]9B). To assess the
   functional significance of this interaction, we silenced Gαi3 using
   targeted shRNA (shGαi3) in priCC-1 cells. In line with the previous
   findings, Gαi3 silencing resulted in a significant decrease in Akt
   phosphorylation (Fig. [207]9C). Importantly, overexpression of GJB5
   using the construct described in Fig. [208]7 failed to rescue Akt
   activation in Gαi3-silenced cells (Fig. [209]9C), indicating that Gαi3
   is essential for GJB5-mediated Akt activation. Overexpression of Gαi3
   in priCC-1 cells led to increased Akt phosphorylation in priCC-1 cells
   (Fig. [210]9D). Significantly, silencing GJB5 using kdGJB5-sh1
   partially inhibited Akt activation induced by Gαi3 overexpression (Fig.
   [211]9D) in priCC-1 cells, suggesting that GJB5 is involved in
   Gαi3-mediated Akt activation. These findings demonstrate that GJB5
   associates with Gαi3 and is required for Gαi3-mediated Akt activation
   in cervical cancer cells. This suggests a novel mechanism by which GJB5
   contributes to Akt activation and cervical cancer progression.

Fig. 9. GJB5 interacts with Gαi3 to promote Akt signaling activation in
cervical cancer cells.

   [212]Fig. 9
   [213]Open in a new tab

   Co-immunoprecipitation (Co-IP) analysis showing interaction between
   GJB5 and Gαi3 proteins in priCC-1 and priCC-2 primary human cervical
   cancer cells (A) or in the listed human cervical cancer tissues (B).
   Western blotting analysis showing the effect of Gαi3 silencing
   (shGαi3), with or without GJB5 overexpression, on Akt phosphorylation
   in priCC-1 cells (C). Western blotting analysis showing the effect of
   Gαi3 overexpression, with or without GJB5 silencing (by kdGJB5-sh1), on
   Akt phosphorylation in priCC-1 cells (D). Data are presented as
   mean ± standard deviation (SD) with n = 5 replicates. Asterisks (*)
   indicate statistically significant differences (P < 0.05) compared to
   the “kdC”/“Vec” treatment. ^# indicates P < 0.05 (D). “N.S.” denotes no
   statistically significant difference (P > 0.05). The experiments were
   independently replicated five times with consistent results.

Silencing of GJB5 impedes cervical cancer xenograft growth in nude mice

   To investigate the functional role of GJB5 in the in vivo growth of
   cervical cancer cells, we established subcutaneous xenograft models in
   nude mice using priCC-1 cells expressing either kdGJB5-sh2 or control
   (kdC) shRNA (see Figs. [214]4–[215]6). Tumor volume was monitored 18
   days after cell injection (labeled as “Day-0”) and over a 42-day
   period. A significant reduction in tumor growth was observed in the
   kdGJB5-sh2 group compared to the control group (Fig. [216]10A). Daily
   growth measurements further corroborated this finding, demonstrating a
   pronounced suppression of tumor expansion in the kdGJB5-sh2 xenografts
   (Fig. [217]10B). At the endpoint (“Day-42”), kdGJB5-sh2 xenografts
   exhibited significantly lower tumor weights compared to control
   xenografts (Fig. [218]10C). No significant changes in body weight were
   observed in either group throughout the experiment (Fig. [219]10D). To
   further elucidate the underlying mechanisms, we analyzed GJB5
   expression in the harvested xenografts, with two xenografts acquired
   for each group. qPCR assays demonstrated a significant reduction in
   GJB5 mRNA levels in kdGJB5-sh2 xenografts (Fig. [220]10E), along with a
   significant downregulation of GJB5 protein (Fig. [221]10F). GJB4
   expression remained unchanged (Fig. [222]10E, F). IHC staining
   confirmed the efficient knockdown of GJB5 protein expression in
   kdGJB5-sh2 xenografts (Fig. [223]10G).

Fig. 10. Silencing of GJB5 impedes cervical cancer xenograft growth in nude
mice.

   [224]Fig. 10
   [225]Open in a new tab

   The priCC-1 primary cervical cancer cells stably transfected with GJB5
   shRNA (kdGJB5-sh2) or control shRNA (kdC) were subcutaneously injected
   into the flanks of female nude mice at a density of seven million cells
   per mouse. Ten mice were included in each experimental group (n = 10).
   Tumor growth was monitored for 42 days, with tumor volume (A, mm³) and
   mouse body weight (D, g) measured every six days starting from day 18
   post injection, designated as “Day-0”. Daily tumor growth rates were
   calculated (B). At day 42, mice were euthanized, and tumors were
   excised and weighed (C). For the subsequent experiments, two xenografts
   from each group (labeled as 1# and 2#) were selected. Tissue lysates
   were prepared for the analysis of GJB5/4 expression levels (E, F), Akt
   and S6K1 phosphorylation/expression levels (H, I), and
   cleaved-caspase-3 and cleaved-PARP1 levels (L). Additionally,
   cytochrome C levels in the tissue lysates were assessed (K).
   Immunohistochemistry (IHC) was performed on tumor sections to assess
   GJB5 protein expression (G) and nuclear Ki-67 counts (J). Additionally,
   TUNEL/DAPI staining was performed in the xenograft sections to evaluate
   apoptosis (M). Data are presented as mean ± standard deviation (SD).
   Statistical significance was determined using a P-value of less than
   0.05 (*P < 0.05) compared to the “kdC” group. “N.S.” indicates
   non-significant differences (P > 0.05). For analyses related to tumor
   volume (A), tumor growth rate (B), tumor weight (C), and mouse body
   weight (D), data were obtained from ten mice per group. For analyses of
   E–M, each xenograft was divided into five segments for individual
   analysis. A scale bar of 100 μm is provided.

   Given the potential involvement of the Akt-mTOR pathway in
   GJB5-mediated cervical cancer cell progression in vitro (see Figs.
   [226]8 and [227]9), we examined the activation status of this pathway.
   Western blotting analysis revealed a significant decrease in the
   phosphorylation levels of Akt and its downstream target, S6K1, in
   kdGJB5-sh2 xenografts compared to controls (Fig. [228]10H, I). A
   significant reduction in Ki-67-positive cells was observed in
   kdGJB5-sh2 xenografts compared to controls (Fig. [229]10J), indicating
   a significant suppression of cancer cell proliferation in vivo. We also
   observed evidence of increased apoptosis in kdGJB5-sh2 xenografts.
   Cytochrome C levels were elevated in the kdGJB5-sh2 xenograft tissues
   compared to controls (Fig. [230]10K). Western blotting analysis
   revealed increased levels of cleaved-caspase-3 and cleaved-PARP1 in
   kdGJB5-sh2 xenografts (Fig. [231]10L). TUNEL staining provided further
   evidence of apoptosis, showing a higher proportion of TUNEL-positive
   nuclei in kdGJB5-sh2 xenografts compared to controls (Fig. [232]10M).
   Collectively, these findings demonstrate that silencing of GJB5 led to
   the inhibition of the Akt-mTOR pathway, induction of apoptosis, and
   suppression of proliferation in priCC-1 xenografts in vivo.

Discussion

   Current therapeutic targets have made significant contributions to the
   management of cervical cancer, yet the limitations of existing
   treatments underscore the critical need for exploring novel targets
   [[233]9, [234]10, [235]12, [236]14, [237]17]. Despite advancements in
   immunotherapy and other targeted therapies, the heterogeneity of
   cervical cancer and the development of resistance to standard
   treatments highlight the urgency of identifying alternative molecular
   pathways [[238]9, [239]10, [240]12, [241]14, [242]17]. Novel
   therapeutic targets can enhance treatment efficacy, reduce side
   effects, and improve patient outcomes by addressing the underlying
   mechanisms driving tumor progression and metastasis [[243]9, [244]10,
   [245]12, [246]14, [247]17]. Furthermore, as the landscape of cervical
   cancer biology continues to evolve, the discovery of new targets may
   facilitate the development of personalized medicine approaches,
   ultimately leading to more effective and tailored therapeutic
   strategies for patients suffering from this disease [[248]9, [249]10,
   [250]12, [251]14, [252]17].

   Our results strongly support GJB5 as a novel therapeutic target for
   cervical cancer based on compelling evidence from multiple analyses.
   Examination of TCGA data revealed a significant increase in GJB5 mRNA
   expression in cervical cancer tissues compared to normal cervical
   epithelium, with high levels of GJB5 correlating with poorer clinical
   outcomes, including reduced overall survival. Furthermore, single-cell
   RNA sequencing confirmed GJB5 overexpression specifically within the
   malignant tumor cell population of cervical squamous cell carcinoma.
   GJB5 expression is also elevated in cervical cancer tissues from
   locally treated patients and in a panel of primary and established
   cervical cancer cells. Functional studies demonstrated that GJB5 shRNA
   or KO not only impaired the viability, proliferation, and migratory
   capacity of cervical cancer cells but also induced apoptotic processes.
   Conversely, forced overexpression of GJB5 led to enhanced malignant
   behaviors. Additionally, the knockdown of GJB5 resulted in a
   significant reduction in the growth of cervical cancer xenografts,
   reinforcing its potential as a promising target for therapeutic
   intervention in cervical cancer management.

   Akt-mTOR signaling hyperactivation is a critical driver of cervical
   cancer malignancy and progression due to its pivotal role in regulating
   cell growth, proliferation, and survival [[253]35, [254]38]. This
   pathway facilitates metabolic reprogramming, allowing cancer cells to
   sustain their energy demands and promote tumorigenesis [[255]35,
   [256]38]. Moreover, hyperactivation of Akt-mTOR signaling has been
   implicated in enhancing resistance to apoptosis, thereby contributing
   to the aggressive nature of cervical cancer [[257]35, [258]38]. The
   pathway also influences the tumor microenvironment by promoting
   angiogenesis and immune evasion, further facilitating cancer
   progression [[259]35, [260]36, [261]43]. Despite the established
   importance of Akt-mTOR signaling in cervical cancer, the precise
   mechanisms underlying its hyperactivation in cervical cancer remain not
   fully understood.

   In our investigations into the underlying mechanisms of cervical cancer
   progression, we showed that GJB5 is integral to the activation of the
   Akt-mTOR signaling pathway. Specifically, GJB5 knockdown or KO resulted
   in a marked reduction in the phosphorylation of both Akt and S6K1,
   indicating a significant impairment of the Akt-mTOR signaling cascade.
   Conversely, GJB5 overexpression was associated with enhanced Akt-mTOR
   signaling in primary human cervical cancer cells. Importantly, in
   GJB5-silenced cervical cancer cells, the restoration of Akt-mTOR
   activation through the expression of caAkt1 reversed the inhibitory
   effects on cell proliferation and migration. This finding underscores
   the critical link between GJB5 and Akt-mTOR signaling in cervical
   cancer malignancy.

   Gαi proteins consist of three main subunits: Gαi1, Gαi2, and Gαi3. Gαi
   proteins are found at elevated levels in various human cancers and play
   a crucial role in tumor development and progression [[262]30–[263]33,
   [264]44]. Recent studies have emphasized the importance of Gαi1 and
   Gαi3 in facilitating signal transmission from multiple receptor
   tyrosine kinases (RTKs), including the epidermal growth factor receptor
   (EGFR), fibroblast growth factor receptor (FGFR), keratinocyte growth
   factor receptor (KGFR), stem cell factor receptor c-kit, brain-derived
   neurotrophic factor receptor TrkB, and vascular endothelial growth
   factor receptor 2 (VEGFR2) [[265]30, [266]39, [267]41, [268]42,
   [269]45, [270]46]. These Gαi proteins are vital for mediating the
   oncogenic Akt-mTOR pathway by these surface receptors [[271]30,
   [272]39, [273]41, [274]42, [275]45, [276]46]. In addition to their
   connections with RTKs, Gαi1 and Gαi3 are also known to interact with
   various non-RTK receptors. Notable examples include the leucine-rich
   repeat-containing G-protein coupled receptor 4 (LGR4) for R-spondin 3
   (RSPO3), CD146 for Netrin-1, the interleukin-4 (IL-4) receptor, and the
   Toll-like receptor 4 (TLR4) for lipopolysaccharide (LPS) [[277]40,
   [278]47–[279]49]. Through these associations, Gαi proteins play a
   significant role in activating the Akt-mTOR signaling pathway,
   underscoring their importance as oncogenic drivers and their potential
   as targets for cancer therapies.

   A recent study identifies Gαi3 as a potential oncotarget in cervical
   cancer, demonstrating that its elevated expression correlates with poor
   patient survival and enhanced cancer cell growth. Silencing or knocking
   out Gαi3 significantly inhibited these processes and reduced Akt-mTOR
   activation, suggesting its role in promoting cervical cancer
   progression [[280]29]. This study identified a novel mechanism by which
   GJB5 promotes Akt signaling in cervical cancer. We demonstrated a
   physical interaction between GJB5 and Gαi3 through co-IP assays in both
   cervical cancer cells and fresh tumor tissues. Silencing Gαi3
   significantly attenuated Akt phosphorylation in cervical cancer cells.
   Importantly, GJB5 overexpression failed to restore Akt activation in
   Gαi3-deficient cells, highlighting the critical role of Gαi3 in
   GJB5-mediated Akt signaling. Conversely, Gαi3 overexpression enhanced
   Akt phosphorylation in primary cervical cancer cells, and this effect
   was partially inhibited by GJB5 silencing. These findings reveal that
   GJB5 promotes Akt activation through a novel mechanism involving its
   interaction with Gαi3 in cervical cancer cells.

Supplementary information

   [281]Original data set^ (1.6MB, pdf)
   [282]Figure S1 and S2^ (1.7MB, pdf)

Acknowledgements