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. 2020 Oct 1;10(10):3138–3156.

SHCBP1 regulates STAT3/c-Myc signaling activation to promote tumor progression in penile cancer

Miao Mo 1, Shiyu Tong 1, Hongling Yin 2, Zhongyuan Jin 2, Xiongbing Zu 1, Xiheng Hu 1
PMCID: PMC7642648  PMID: 33163262

Abstract

A challenge in developing novel strategies for penile cancer (PC) is the limited understanding of the regulatory mechanisms involved in PC development. This study aims to examine the expression of SHC SH2 Domain-Binding Protein 1 (SHCBP1) in PC and to explore its oncogenic function. Aberrant SHCBP1 expression was observed in PC tissues compared with normal penile tissues. SHCBP1 expression was significantly associated with the pathological grade, T stage, nodal status, and pelvic lymph node metastasis, and could serve as an independent factor for unfavorable overall survival in PC. Manipulation of SHCBP1 expression affected cell proliferation, soft agar clonogenesis, and cell migration and invasion in PC cell lines. Moreover, we identified STAT3/c-Myc signaling as a potential downstream target of SHCBP1. SHCBP1 interacted with JAK2 and STAT3 upon EGF stimulation, which might regulate STAT3/c-Myc signaling activation in PC cells. Disruption of STAT3/c-Myc signaling attenuated cell proliferation and cell migration/invasion in PC cell lines. Nevertheless, overexpression of constitutively activated STAT3 or c-Myc rescued cell proliferation and cell migration/invasion caused by SHCBP1 depletion in PC cell lines. Consistently, SHCBP1 depletion attenuated STAT3/c-Myc signaling and suppressed tumor growth in a murine xenograft model. Importantly, correlated expression of SHCBP1, p-STAT3, and c-Myc was observed in PC tissues, confirming the clinical relevance of SHCBP1/STAT3/c-Myc signaling in PC. In conclusion, aberrant SHCBP1 expression could serve as a potential prognostic biomarker for PC. SHCBP1 might activate the STAT3/c-Myc signaling pathway to promote tumor progression in PC, which may serve as a potential target for PC treatment.

Keywords: Penile cancer, SHCBP1, STAT3, c-Myc, prognosis, tumor progression

Introduction

Penile cancer (PC) is a rare malignancy in the United States (0.5-1.6 per 100,000 men), with a significantly higher incidence-up to 20-30 times greater- in some regions of Africa, Asia, and South America [1]. Currently, a surgical operation is recommended as the major treatment for PC. Besides the surgical approach, brachytherapy, chemotherapy, and targeted therapy have also been applied in the clinical management of PC [2,3]. Although patients with early-stage disease have an estimated 10-year survival of 89%, patients with advanced disease have only an estimated survival of 21% at two years [4]. The clinical outcomes of PC are associated with tumor grade, pathological subtype, human papillomavirus (HPV) status, and clinical stage, with the presence of inguinal nodal metastases being the most important prognostic factor for patient survival [5]. Despite considerable progress in clinical treatment, the survival of patients with PC has not improved in the past 20 years [6].

A challenge in developing novel strategies for PC is the limited understanding of the heterogeneity of the oncogenic drivers of disease. Although HPV infection is a significant contributing factor for PC [7], an extended list of mechanisms responsible for PC has also been documented. Genomic profiling identified clinically relevant genomic alterations of oncogenes and tumor suppressors, with TP53, CDKN2A, PIK3CA, and HRAS among the most frequently altered genes [8]. Nevertheless, hyperactivation of oncogenic pathways such as EGFR/RAS, Wnt/β-Catenin, and PI3K/AKT/mTOR, dysregulated DNA methylation, and overexpression of miRNAs were also shown to be involved in PC development [9-12]. Recently, we showed that ID1 and CEACAM19 are closely connected to tumor progression in patients with PC [13,14]. Despite this aforementioned progress in the PC study, there is still a limited understanding of the biological regulators of prognostic and therapeutic importance in penile cancer. Further knowledge on the molecular mechanisms underlying PC progression may help in the development of therapeutic strategies.

SHC SH2 Domain-Binding Protein 1 (SHCBP1) was initially identified as a potential gene associated with cell proliferation in fibroblasts [15,16]. SHCBP1 exerts a crucial physiological function in normal tissue development. Chen et al. showed that SHCBP1 is an essential component of FGF signaling in neural progenitor cells [17]. SHCBP1 is also upregulated during T cell proliferation and regulates CD4+ T cell effector function in vivo [18]. Recently, SHCBP1 was shown to be closely associated with cancer development. Aberrant SHCBP1 expression is associated with poor prognosis and aggressive phenotype in gastric, prostate, and breast cancer [19-24]. However, the expression and oncogenic role of SHCBP1 in PC remain mostly unknown. Here, we sought to determine the association between SHCBP1 expression and clinicopathological characteristics of PC and to explore its oncogenic function in PC tumorigenesis.

Materials and methods

Patient and tumor characteristics

Archival paraffin-embedded PC tissues (n = 105) were collected for immunohistochemistry. The PC patients included in this study had complete patient and tumor characteristics, and underwent surgery for PC between 2012 and 2014 at Xiangya Hospital, Central South University. Patients with known chemotherapy or brachytherapy before the surgery were excluded from the study. TNM staging was assigned based on the American Join Committee on Cancer, 8th edition [25]. The study protocols were approved by the research ethics committee in Xiangya hospital (Rev No. 201805847) with informed consent obtained from all patients. Cancer and vital status were determined by clinical follow-up at our institution.

Reagents and cell lines

Primary antibody against SHCBP1 was kindly provided by Proteintech (Rosemont, IL, USA). Primary antibodies against p-STAT3 (Tyr705), STAT3, c-Myc, JAK2, cleaved caspase-3 and β-actin were obtained from Cell Signaling Technology (Beverly, MA USA); Human penile cancer cell lines Penl1, Penl2, 149RCa and LM156 were kindly provided by Prof. Hui Han (Department of Urology, Cancer hospital, Sun Yat-sen University) [26]. These cell lines was cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 4 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Lentiviral plasmids expressing shRNAs targeting SHCBP1, STAT3, or c-Myc were purchased from Genecopoeia Inc. (Rockville, MD, USA). Lentiviral LV105 plasmids expressing empty vector (EV), c-Myc or SHCBP1 were purchased from Genecopoeia Inc. (Rockville, MD, USA). Lentiviral plasmids Flag-tagged STAT3DN (dominant negative, #24984) and STAT3CA (constitutively active, #24983) were provided by Addgene (Watertown, MA, USA). The packaging procedure for lentiviral particles was conducted as we described previously [13,14].

Cell growth analysis

Cell growth was measured by CCK-8 assay as we described previously [13]. The CCK-8 absorbance (OD450) was measured with a MK3 microplate reader (Thermo Scientific, USA).

Soft agar assay

Soft agar assay was used to examine the in vitro clonogenesis of PC cells as described previously [27]. Briefly, the 2 mL culture medium with 0.5% agar was first plated into each well of a 6 cm culture dish. After the agar solidified, each well received another 2 mL of 0.35% agar in culture medium containing 2 × 104 cells. After 2 weeks, colonies were counted. The number of colonies was determined microscopically by manually counting from triplicate wells for each cell line.

Wound healing assay

Cell migration ability was measured by wound healing assay as we described previously [14]. Briefly, after PC cells were grown fully confluent, a uniform scratch was made for each experiment group. The distance between the wound sides was measured immediately after the scratch or after indicated time interval.

Transwell invasion assay

Cell invasion assay were performed based on transwell chamber with 8 μm pores as described previously [28]. Briefly, PC cells were suspended in DMEM medium at 5 × 105 cells/ml. Each transwell of 24-well plate was pre-coated with 50 μl Matrigel. RPMI 1640 medium (600 μl) containing 10% FBS was added to each well (lower compartment), and 0.1 ml (0.5 × 105 cells) of cell suspension was added onto each transwell insert (upper compartment). The plates were incubated for 24 h at 37°C. The invaded cells on the bottom surface of the membrane were fixed by 4% paraformaldehyde, and stained by 0.2% crystal violet solution (Sigma-Aldrich, USA). After the wash, cells were photographed with Olympus BX43 microscope. Then the stained cells were eluted by 20% glacial acetic acid and measured with MK3 microplate reader (Thermo Scientific, USA) at 570 nm.

Western blotting

Cell lysates were prepared with RIPA cell lysis buffer. The experimental procedure of Western blotting was conducted as we described previously [13]. Protein blots were visualized using the ECL system (Abcam, Cambridge, MA, USA).

Luciferase reporter assay

Luciferase reporter assay was conducted to examine cancer-related pathway activity as described previously [27]. Briefly, PC cells were grown in 96-well plate and transfected with plasmid construct of transcription factor-responsive reporter of each pathway. After 48 hours, the changes in expression of each pathway in cells with or without SHCBP1 knockdown were determined by measuring the generated firefly and Renilla luminescent signals using the Dual-Glo Luciferase Assay system (Promega, Madison, WI, USA). The luciferase activity of scramble shRNA control was regarded as 100%.

Co-immunoprecipitation

Co-immunoprecipitation of SHCBP1 or JAK2 from PC cell lysates was carried out as described previously [27]. SHCBP1 and JAK2 were immunoprecipitated using a rabbit anti-SHCBP1 or anti-JAK2 antibody. Rabbit IgG were used as negative controls. Immune complexes were captured using the Protein G Dynabeads (Beyotime Biotechnology), and immunoprecipitates were analyzed by SDS-PAGE followed by immunoblotting with mouse monoclonal anti-SHCBP1 antibody (Abnova) and mouse monoclonal anti-STAT3 or anti-JAK2 antibody (Cell Signaling). Immunoreactive bands were visualized using enhanced chemiluminescence.

Immunohistochemistry (IHC)

Immunohistochemistry on PC tissue sections (n = 105) were performed as we described previously [13,14]. Briefly, the sections were dewaxed in xylene, rehydrated with graded alcohols and subjected to heat induced epitope retrieval. The sections were then incubated overnight at 4°C with primary antibodies (dilution: SHCBP1: 1:200; p-STAT3: 1:100; c-Myc: 1:200; Ki-67: 1:500), followed by incubation with a horseradish peroxidase conjugated goat anti rabbit secondary antibody (DAKO, Denmark) for 30 min at room temperature. Antigen-antibody reactions were visualized by exposure to 3,3-diaminobenzidine and hydrogen peroxide chromogen substrate (DAKO, Denmark). The staining positivity ≥ 30% was regarded as high expression [13,14]. IHC results were examined and scored by two pathologists (H.Y and Z.J).

RNA sequencing

RNA sequencing was conducted to identify gene mRNA expression in Penl2 cells transfected with shRNAs targeting SHCBP1 or STAT3. The mRNA libraries were constructed using NEB Next Ultra II Directional RNA Library Prep Kit (Ipswich, MA, USA). High-throughput sequencing was performed on an Illumina HiSeq 4000 platform. Expression levels of each protein-coding gene were expressed as Fragments per kilobase of transcript per million fragments mapped (FPKM). The analysis of differentially expressed genes (DEGs) is performed with the Limma package [29]. Genes meeting the cutoff criteria (adjusted P < 0.05 and |log2FC| ≥ 1) are reported as DEGs.

Pathway and process enrichment analysis

Pathway and process enrichment analysis was conducted in Metascape (http://metascape.org/gp/index.html) with ontology sources for GO Biological Processes and KEGG Pathway [30].

GEO dataset

GEO dataset GSE57955 could be downloaded from NCBI GEO website (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57955). Gene expression data were analyzed as described previously [31]. Genes with a mean log2 signal ratio (penile cancer/normal penile tissue pool) of ≥ 2.0 and ≤ -2.0 were considered differentially expressed.

Gene set enrichment analysis (GSEA)

GSE57955 dataset was used for GSEA analysis. Gene expression profiles were compared between SHCBP1-high (n = 20) and SHCBP1-low PC (n = 19) based on enrichment of KEGG pathway signatures [32]. The nominal p value (NOM p-val) and false discovery rate (FDR)-corrected q value (FDR q-val) are indicated. A nominal P value of < 0.05 was considered significant in this analysis.

Animal studies

Immunocompromised nude mice were obtained from the breeding facility at the animal center of Central South University. All animal studies were performed in accordance with institutional ethical guidelines for experimental animal care. In xenograft study, 1 × 106 Penl2 cells were subcutaneously inoculated into the flank of each nude mice (n = 6). In order to determine tumor volume by external caliper, the greatest longitudinal diameter (a) and the greatest transverse diameter (b) were determined. Tumor volume based on caliper measurements was calculated by the modified ellipsoidal formula: tumor volume (mm3) = a × b2/2. Mice were sacrificed 22 days after cell inoculation, and the subcutaneous xenografts were removed, washed by phosphate buffered saline (PBS), and weighted. The expression of SHCBP1, p-STAT3, c-Myc, and Ki-67 in xenograft tissues were evaluated by western blotting and immunohistochemistry.

Statistical analysis

The statistical software package SPSS 16.0 was used in this study. Chi-square test was conducted to analyze the relationship between SHCBP1 expression and clinicopathological parameters. Kaplan-Meier curves of overall survival were plotted and survival in the groups was compared by log-rank test. Multivariable Cox regression analysis was conducted to identify the prognostic factors that influence overall survival. Significance between two groups was performed using Student’s t test. Significance between more than two groups was evaluated by one way analysis of variance (ANOVA). A two-tailed P < 0.05 was considered significant in all tests.

Results

SHCBP1 is highly expressed in PC tissues

The mRNA expression of SHCBP1 in PC was analyzed based on GEO databases (GSE57955). As shown in Figure 1A, 87.2% of PC tissues (34/39) exhibited a high level of SHCBP1 expression (PC vs. normal penile tissue pool (NPT), Log2 PC/NPT ≥ 2). SHCBP1 expression did not differ between HPV+ and HPV- PC tissues (P > 0.05, Figure 1B). The expression of SHCBP1 protein in normal penile tissues (n = 30) and PC tissues (n = 105) was assessed by immunohistochemistry. SHCBP1 staining was low or absent in normal penile tissues (Figure 1C, left panel). Overall, 38.1% (40/105) of PC cases exhibited high SHCBP1 expression (staining positivity ≥ 30%) (Figure 1C, right panel). The association between SHCBP1 and clinicopathological features of the PC cohort is summarized in Table 1. As shown in Table 1, high SHCBP1 expression was significantly related to pathological grade (P = 0.032), T stage (P = 0.017), lymph node metastasis (P = 0.003), and pelvic lymph node metastasis (pelvic LNM) (P = 0.019) but not to age (P = 0.632), phimosis (P = 0.965), body mass index (P = 0.333), or histological subtype (P = 0.266). Survival analysis showed that high SHCBP1 expression was associated with shorter overall survival in our PC cohort (Figure 1D). We also conducted univariate and multivariate Cox regression analysis, and the results are summarized in Table 2. Univariate Cox regression analysis results showed that pathological grade (P = 0.025), T stage (P = 0.006), nodal status (P < 0.001), pelvic LNM (P < 0.001), and high SHCBP1 expression (P < 0.001) were prognostic factors for overall survival in our PC cohort (Table 2). However, multivariate Cox regression analysis indicated that the nodal status (P < 0.001; HR = 8.056), pelvic LNM (P < 0.001; HR = 24.437), and high SHCBP1 expression (P = 0.025; HR = 3.106) could serve as independent prognostic factors for unfavorable overall survival (Table 2). Western blotting showed that SHCBP1 exhibited differential expression patterns in normal penile tissues (NPT1, NPT2) and a panel of PC cell lines (Penl1, Penl2, 149RCa, and LM156), with high SHCBP1 expression seen in Penl1 and Penl2 cells (Figure 1E).

Figure 1.

Figure 1

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SHCBP1 is highly expressed in PC and associated with unfavorable prognosis. A. Waterfall plot of relative SHCBP1 expression in GSE57955 dataset (n = 39). Relative SHCBP1 expression was calculated with reference to normal penile tissue pool. B. SHCBP1 expression was not associated with HPV status in GSE57955 dataset. HPV+ cases vs. HPV- cases, P > 0.05. C. SHCBP1 was highly expressed in PC tissues (n = 105) compared to normal penile tissues (n = 30). Representing micrographs showed SHCBP1 expression in normal penile tissues or PC tissues, respectively. Bars: 100 μm. D. High SHCBP1 expression was associated with unfavorable overall survival in our PC cohort. E. Expression of SHCBP1 in normal penile tissues (NPT1, NPT2) and a panel of PC cell lines (Penl1, Penl2, LM156, l49RCa). β-Actin was used as loading control.

Table 1.

Association between clinicopathologic characteristics and SHCBP1 expression in PC cohort

Clinicopathological Parameters SHCBP1 low expression SHCBP1 High expression Odds Ratio P value
Age (year) 1.212 0.632
    < 54 34 (53.3%) 19 (47.5%)
    ≥ 54 31 (46.7%) 21 (52.5%)
Phimosis 1.021 0.965
    Yes 49 (75.4%) 30 (75.0%)
    No 16 (24.6%) 10 (25.0%)
Body mass index 1.500 0.333
    < 24 45 (69.2%) 24 (60.0%)
    ≥ 24 20 (30.8%) 16 (40.0%)
Pathological grade 2.421 0.032*
    G1 46 (70.8%) 20 (45.0%)
    G2 + G3 19 (29.2%) 20 (55.0%)
Histological subtype 0.609 0.266
    Usual 42 (64.6%) 30 (75.0%)
    Others 23 (35.4%) 10 (25.0%)
T stage 2.667 0.017*
    T1 40 (61.5%) 15 (37.5%)
    T2 + T3 25 (38.5%) 25 (62.5%)
Nodal status 3.451 0.003*
    Negative 48 (77.5%) 18 (45.0%)
    Positive 17 (22.5%) 22 (55.0%)
Pelvic LNM 9.143 0.019*
    No 64 (98.5%) 35 (87.5%)
    Yes 1 (1.5%) 5 (12.5%)
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*

P < 0.05.

Table 2.

Univariate and multivariate Cox proportional hazard model for clinicopathological parameters affecting overall survival in PC cohort

Clinicopathological parameters Univariate analysis Multivariate analysis
HR (95% CI) P value HR (95% CI) P value
Age (< 54 vs. ≥ 54) 0.266
Subtype (Usual vs. others) 0.192
Grade (G2 + G3 vs. G1) 2.613 (1.130-6.043) 0.025 0.062
T stage (T2 + T3 vs. T1) 3.718 (1.465-9.435) 0.006 0.350
Nodal status 6.257 (2.461-15.911) < 0.001 8.056 (2.760-23.511) < 0.001
Pelvic LNM 19.821 (7.180-54.715) < 0.001 24.437 (6.489-92.021) < 0.001
High SHCBP1 expression 5.633 (2.217-14.311) < 0.001 3.106 (1.156-8.344) 0.025
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Knockdown of SHCBP1 expression suppresses malignant phenotypes in SHCBP1-high PC cell lines

We sought to investigate further the oncogenic function of SHCBP1 in SHCBP1-high Penl1 and Penl2 cells. These two cell lines were transfected with non-targeting scrambled (Scr) or specific shRNAs targeting SHCBP1 (sh-1 and sh-2). As shown in Figure 2A, SHCBP1 expression was greatly reduced by shSHCBP1 compared with Scr in Penl1 and Penl2 cells. We next examined the effect of SHCBP1 expression on the cell growth of PC cells by CCK-8 assay, and the results showed that shSHCBP1-transfected PC cells grew slower than those transfected with Scr shRNA (P < 0.05; Figure 2B). Caspase-3 activities were increased in SHCBP1 knockdown groups compared with Scr control groups (P < 0.05; Figure 2C). Moreover, soft agar clonogenesis of PC cells in shSHCBP1 groups decreased greatly, compared with Scr groups (P < 0.05; Figure 2D). We also observed a significantly retarded wound-healing rate in PC cells expressing shSHCBP1 shRNAs compared with those expressing in the Scr control (P < 0.05; Figure 2E). Transwell invasion assay was also performed to examine the effects of SHCBP1 on cell invasion. As shown in Figure 2F, knockdown of SHCBP1 expression attenuated the invasion of Penl1 and Penl2 cells compared with Scr control (P < 0.05). As both SHCBP1 shRNAs demonstrated a similar effect on PC cells, SHCBP1 sh-2 shRNA was used for further experiments. In addition, the cell viability attenuated by SHCBP1 sh-2 shRNA could be mostly restored by transfection of recombined shRNA-resistant SHCBP1 plasmid, suggesting that the off-target effect of sh-2 shRNA might be minimal (Figure S1A, S1B).

Figure 2.

Figure 2

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Knockdown of SHCBP1 impaired the malignant phenotypes in SHCBP1-high PC cell lines. A. SHCBP1 expression was greatly reduced by lentivirus-mediated shRNA knockdown compared to scramble (Scr) control in Penl1 and penl2 cells. β-Actin was used as loading control. B. SHCBP1 knockdown attenuated cell proliferation in Penl1 and Penl2 cells. The cell viability in Scr control was regards as 100%. n = 4, *P < 0.05, as compared with Scr control. C. SHCBP1 knockdown induced caspase-3 activity in Penl1 and Penl2 cells. The caspase-3 activity in Scr control was regards as 100%. n = 3, *P < 0.05, as compared with Scr control. D. SHCBP1 knockdown inhibited soft agar clonogenesis in Penl1 and Penl2 cells. The soft agar clonogenesis in Scr control was regards as 100%. n = 3, *P < 0.05, as compared with Scr control. Bars: 100 μm. E. Knockdown of SHCBP1 expression inhibited cell migration of PC cells. Bars: 100 μm. The cell migration in Scr control was regards as 100%. n = 3, *P < 0.05. F. Knockdown of SHCBP1 expression inhibited transwell invasion of PC cells. Bars: 100 μm. The cell invasion in Scr control was regards as 100%. n = 3, *P < 0.05.

Overexpression of SHCBP1 expression promotes malignant phenotypes in SHCBP1-low PC cell lines

The function of SHCBP1 was also explored in the SHCBP1-low PC cell line 149RCa and LM156. As shown in Figure 3A, SHCBP1 expression was significantly increased in 149RCa and LM156 cells transfected with SHCBP1 lentivirus compared with the empty vector (EV) control. Next, we examined the effect of SHCBP1 expression on the cell growth of PC cells by CCK-8 assay. SHCBP1-expressing PC cells grew faster than those transfected with EV (P < 0.05; Figure 3B). Caspase-3 activities were decreased in SHCBP1 groups as compared with that in EV groups (P < 0.05; Figure 3C). Moreover, soft agar clonogenesis of PC cells in SHCBP1 groups was significantly increased compared with that in the EV groups (P < 0.05; Figure 3D). Further, a significantly accelerated wound-healing rate in PC cells expressing SHCBP1 was observed, compared with those expressing in EV control (P < 0.05; Figure 3E). Transwell invasion analysis showed that overexpression of SHCBP1 enhanced the invasion of 149RCa and LM156 cells compared with that in EV control (Figure 3F, P < 0.05).

Figure 3.

Figure 3

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Over-expression of SHCBP1 enhances the malignant phenotypes in SHCBP1-low PC cell lines. A. SHCBP1 expression was considerably increased by constitutive SHCBP1 expression compared to empty vector (EV) control in LM156 and 149RCa cells. β-Actin was used as loading control. B. Constitutive SHCBP1 expression promoted cell proliferation in LM156 and 149RCa cells. The cell viability in EV control was regards as 100%. n = 4, *P < 0.05, as compared with EV control. C. Over-expression of SHCBP1 attenuated caspase-3 activity in LM156 and 149RCa cells. The caspase-3 activity in EV control was regards as 100%. n = 3, *P < 0.05, as compared with EV control. D. Constitutive SHCBP1 expression enhanced soft agar clonogenesis in LM156 and 149RCa cells. The soft agar clonogenesis in EV control was regards as 100%. n = 3, *P < 0.05, as compared with EV control. Bars: 100 μm. E. Over-expression of SHCBP1 expression promoted cell migration of PC cells. Bars: 100 μm. The cell migration in EV control was regards as 100%. n = 3, *P < 0.05. F. Over-expression of SHCBP1 enhanced transwell invasion of PC cells. Bars: 50 μm. The cell invasion in EV control was regards as 100%. n = 3, *P < 0.05.

SHCBP1 regulates the STAT3/c-Myc signaling pathway in PC cells

A luciferase reporter assay on cancer-related signaling pathways was conducted to identify downstream signaling of SHCBP1 in PC cells. As shown in Figure 4A, knockdown of SHCBP1 led to a dramatic decline of luciferase reporter activity in STAT3 and c-Myc pathways. A considerable reduction of p-STAT3 and c-Myc levels was observed in SHCBP1-depleted Penl1 and Penl2 cells compared with Scr control (Figure 4B), whereas overexpression of SHCBP1 increased p-STAT3 and c-Myc levels in 149RCa and LM156 cells (Figure 4C). We conducted RNA sequencing analysis on SHCBP1- or STAT3-depleted Penl2 cells (Table S1). A number of up-regulated (Log2FC ≥ 1, n = 22) and down-regulated (Log2FC ≤ -1, n = 99) genes were commonly identified in shSHCBP1 and shSTAT3 group (Figure S2A, S2B). Notably, important genes involved in oncogenic function (Myc, SOX9, FOXA1, SNAI), cell cycle regulation (CCND1, CCNA1, PLK3), apoptosis (MCL1, BCL2L1, BAX), and angiogenesis/inflammation (VEGFC, IL6, CXCL8, CCL20, IL1A) were differentially expressed in shSHCBP1 and shSTAT3 group (Figure 4D). Pathway and process enrichment analysis showed that the Go biological processes (cytokine-mediated signaling pathway, positive regulation of response to external stimulus, response to virus, regulation of cell death, and cellular response to lipid) and KEGG pathways (transcriptional misregulation in cancer, bladder cancer, IL-17 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and JAK-STAT signaling pathway) were highly enriched in the down-regulated genes in shSHCBP1 and shSTAT3 group (Figure 4E). In contrast, the up-regulated genes were shown to enrich Go biological processes (post-embryonic development, appendage morphogenesis, positive regulation of apoptotic signaling pathway, cellular response to TGF-β stimulus) and KEGG pathways (HTLV-1 infection, pathways in cancer) (Figure 4E). Western blotting analysis showed that knockdown of SHCBP1 or STAT3 reduced anti-apoptosis genes (MCL1, BCL-XL) and induced pro-apoptosis genes (BAX, cleaved caspase-3) in Penl1 and Penl2 cells (Figure S2C).

Figure 4.

Figure 4

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SHCBP1 regulates downstream STAT3/c-Myc signaling in PC cells. A. Multiple pathway reporter analysis identified potential cancer-related pathways regulated by SHCBP1. B. Western blotting analysis on STAT3/c-Myc signaling following SHCBP1 depletion in Penl1 and Penl2 cells. C. Western blotting analysis on STAT3/c-Myc signaling following over-expression of SHCBP1 in LM156 and 149RCa cells. D. Heatmap showing differential expression of important genes involved in oncogenic function, cell cycle regulation, apoptosis, and angiogenesis/inflammation in SHCBP1- or STAT3-depleted Penl2 cells. E. Enrichment of KEGG pathways and GO biological processes in common DEGs in SHCBP1- or STAT3-depleted Penl2 cells.

JAK2/STAT3 signaling regulated by SHCBP1 might be driven by EGFR

Immunoprecipitation analysis showed that SHCBP1 interacts with STAT3 and its upstream kinase JAK2 in Penl1 and Penl2 cells (Figure 5A). However, the interaction between JAK2 and STAT3 was greatly disrupted by SHCBP1 knockdown in Penl1 and Penl2 cells (Figure 5B). STAT3 is an essential signaling protein engaged by EGFR to promote cellular growth, differentiation, and survival in cancer cells [33]. To examine the effect of EGFR signaling on SHCBP1-regulated JAK2/STAT3 activation, we treated serum-deprived Penl2 cells with EGF (50 ng/ml) for 6 h. As shown in Figure 5C, the interaction between SHCBP1 and JAK2:STAT3 was considerably induced by EGF. Next, we treated serum-deprived Penl2 cells (Scr or shSHCBP1) with EGF (50 ng/ml) or EGFR inhibitor erlotinib. Western blotting analysis showed that EGF stimulation increased p-EGFR, p-STAT3, and c-Myc expression in SHCBP1-expressing Penl2 cells, which was blocked by EGFR inhibitor erlotinib; SHCBP1 depletion led to decreased expression of p-STAT3 and c-Myc in EGF-treated Penl2 cells compared with Scr control (Figure 5D). Therefore, the JAK2/STAT3 signaling regulated by SHCBP1 might be driven by EGFR.

Figure 5.

Figure 5

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A. SHCBP1 interacts with JAK2 and STAT3 in Penl1 and Penl2 cells. Rabbit anti-SHCBP1 antibody was used to immunoprecipitate SHCBP1 protein. Rabbit IgG was used as negative control. B. SHCBP1 might regulate the interaction between JAK2 and STAT3 in Penl1 and Penl2 cells. Rabbit anti-JAK2 antibody was used to immunoprecipitate STAT3 proteins. Rabbit IgG was used as negative control. C. SHCBP1 interacts with JAK2 and STAT3 upon EGF stimulation. After serum deprivation for 36 hours, Penl2 cells were stimulated with EGF (50 ng/ml) for 6 hours. SHCBP1 antibody was used to immunoprecipitate SHCBP1 protein. Rabbit IgG was used as negative control. D. SHCBP1 depletion attenuates EGF-induced STAT3/c-Myc signaling activation in Penl2 cells. After serum deprivation for 36 hours, PC cells (Scr or shSHCBP1) were stimulated with EGF (50 ng/ml) alone or EGF plus erlotinib (2 μM) for 6 hours. Western blotting was conducted to detect the protein levels of STAT3/c-Myc signaling components.

Knockdown of STAT3 or c-Myc attenuates malignant phenotypes in SHCBP1-high PC cell lines

To verify the function of STAT3/c-Myc signaling in PC, we depleted STAT3 or c-Myc expression in Penl1 and Penl2 cells (Figure 6A). The knockdown of STAT3 reduced c-Myc expression in Penl1 and Penl2 cells, suggesting that c-Myc is a downstream target gene of STAT3 (Figure 6A). Next, we examined the cellular function of STAT3/c-Myc in Penl1 and Penl2 cells. As shown in Figure 6B, shSTAT3, or shc-Myc-transfected PC cells grew slower than those transfected with Scr shRNA (P < 0.05; Figure 6B). Caspase-3 activities were increased in STAT3 or c-Myc knockdown groups compared with that in Scr control groups (P < 0.05; Figure 6C). Moreover, soft agar clonogenesis of PC cells in the shSTAT3 or shc-Myc group decreased greatly as compared with that in the Scr group (P < 0.05; Figure 6D). We also observed a significantly retarded wound-healing rate in PC cells expressing the shSTAT3 or shc-Myc groups compared with those expressing in the Scr control (P < 0.05; Figure 6E). Transwell invasion assay was also performed to examine the effects of shSTAT3 or shc-Myc on cell invasion. As shown in Figure 6F, knockdown of STAT3 or c-Myc expression attenuated the invasion of Penl1 and Penl2 cells compared with that in Scr control (P < 0.05).

Figure 6.

Figure 6

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Knockdown of STAT3 or c-Myc impaired the malignant phenotypes in SHCBP1-high PC cell lines. A. STAT3 or c-Myc expression was reduced by lentivirus-mediated shRNA knockdown compared to scramble (Scr) control in Penl1 and penl2 cells. β-Actin was used as loading control. B. STAT3 or c-Myc knockdown attenuated cell proliferation in Penl1 and Penl2 cells. The cell viability in Scr control was regards as 100%. n = 4, *P < 0.05, as compared with Scr control. C. STAT3 or c-Myc knockdown induced caspase-3 activity in Penl1 and Penl2 cells. The caspase-3 activity in Scr control was regards as 100%. n = 3, *P < 0.05, as compared with Scr control. D. STAT3 or c-Myc knockdown inhibited soft agar clonogenesis in Penl1 and Penl2 cells. The soft agar clonogenesis in Scr control was regards as 100%. n = 3, *P < 0.05, as compared with Scr control. Bars: 100 μm. E. Knockdown of STAT3 or c-Myc expression inhibited cell migration of PC cells. Micrographs showed the results of Penl2 cells. Bars: 100 μm. The cell migration in Scr control was regards as 100%. n = 3, *P < 0.05. F. Knockdown of STAT3 or c-Myc expression inhibited transwell invasion of PC cells. Bars: 50 μm. The cell invasion in Scr control was regards as 100%. n = 3, *P < 0.05.

Overexpression of constitutively activated STAT3 or c-Myc partly restores malignant phenotypes in SHCBP1-depleted PC cells

STAT3CA (constitutively active), STAT3DN (dominant-negative), or c-Myc was constitutively expressed in SHCBP1-depleted Penl1 and Penl2 cells in order to confirm the hypothesis that STAT3/c-Myc signaling is required for SHCBP1 function in PC. As shown in Figure 7A, overexpression of STAT3CA or c-Myc restored c-Myc expression in SHCBP1-depleted Penl1 and Penl2 cells. The cellular function of STAT3 mutants and c-Myc was analyzed in Penl1 and Penl2 cells. As shown in Figure 7B, in contrast to the empty vector (EV), overexpression of STAT3CA or c-Myc, but not STAT3DN, rescued cell proliferation attenuated by SHCBP1 knockdown in Penl1 and Penl2 cells. Soft agar clonogenesis of PC cells in the STAT3CA or c-Myc group was greatly increased compared with that in the EV group (Figure 7C). Moreover, caspase-3 activities induced by SHCBP1 knockdown were markedly reduced by the constitutive expression of STAT3CA or c-Myc (Figure 7D; P < 0.05). The wound-healing rate of PC cells was partly restored in cells expressing STAT3CA or c-Myc following SHCBP1 knockdown (Figure 7E; P < 0.05). Transwell invasion analysis showed that overexpression of STAT3CA or c-Myc promoted cellular invasiveness compared with EV in SHCBP1-depleted Penl1 and Penl2 cells (Figure 7F; P < 0.05).

Figure 7.

Figure 7

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Over-expression of constitutively active STAT3 or c-Myc rescued malignant phenotypes impaired by SHCBP1 depletion. A. Over-expression of STAT3CA (constitutively active), STAT3DN (dominant negative) or c-Myc in SHCBP1-depleted Penl1 and Penl2 cells. B. Over-expression of STAT3CA or c-Myc rescued cell proliferation suppressed by SHCBP1 depletion. The cell viability in Scr control was regards as 100%. n = 4, *P < 0.05, as compared with EV. C. Over-expression of STAT3CA or c-Myc rescued soft agar clonogenesis inhibited by SHCBP1 depletion. The soft agar clonogenesis in Scr control was regards as 100%. n = 4, *P < 0.05, as compared with EV. D. Over-expression of STAT3CA or c-Myc reduced caspase-3 activity induced by SHCBP1 depletion. The caspase-3 activity in Scr control was regards as 100%. n = 3, *P < 0.05, as compared with EV. E. Over-expression of STAT3CA or c-Myc rescued cell migration inhibited by SHCBP1 depletion. Bars: 100 μm. The cell migration in Scr control was regards as 100%. n =3, *P < 0.05, as compared with EV. F. Over-expression of STAT3CA or c-Myc rescued cell invasion inhibited by SHCBP1 depletion. Bars: 50 μm. The cell invasion in Scr control was regards as 100%. n = 3, *P < 0.05, as compared with EV.

Knockdown of SHCBP1 attenuates STAT3/c-Myc signaling and suppresses tumor growth in a PC xenograft model

The in vitro findings that knockdown of SHCBP1 suppressed the malignant phenotypes of PC cell lines guided the following in vivo studies. The effect of SHCBP1 depletion was examined in nude mice with Penl2 subcutaneous xenografts. The group of mice inoculated with Penl2/Scr PC cells grew subcutaneous tumors more rapidly than those with Penl2/shSHCBP1 cells (P < 0.05; Figure 8A). The tumor weight in the shSHCBP1 group was significantly smaller than that in the Scr group (Figure 8B). Western blotting analysis showed that SHCBP1 depletion attenuated p-STAT3 and c-Myc expression and induced cleaved caspase-3 in the xenografts (Figure 8C). Immunohistochemical staining showed that the shSHCBP1 group exhibited decreased SHCBP1, p-STAT3, c-Myc, and Ki-67 expression in the tumors, compared with the Scr control group (Figure 8D). Therefore, SHCBP1 might serve as a potential therapeutic target for PC.

Figure 8.

Figure 8

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Targeting SHCBP1 suppressed in vivo tumor growth and disrupted STAT3/c-Myc signaling. A. SHCBP1 depletion attenuated subcutaneous xenograft growth. Tumor volume was measured every two days after Penl2 inoculation. n = 6, *P < 0.05, Scr control vs. shSHCBP1. B. Subcutaneous Penl2 xenografts were taken out at day 22, photographed and weighted. P = 0.008, Scr control vs. shSHCBP1. C. Western blotting analysis on protein lysates extracted from Penl2 xenografts with or without SHCBP1 depletion. D. Immunohistochemical staining on Penl2 xenografts with or without SHCBP1 depletion at day 22 after implantation. The tissue sections were incubated with antibodies against indicated antibodies (SHCBP1, p-STAT3, c-Myc, and Ki-67). Diaminobenzidine (DAB) was used as a chromogen, followed by counterstaining with hematoxylin. Bar, 50 μm.

High SHCBP1 expression significantly correlates with STAT3/c-Myc signaling activation in PC

Given the strong effect of SHCBP1 on PC cell lines, we sought to explore the correlation between SHCBP1 expression and STAT3/c-Myc signaling activation in clinical PC specimens by immunohistochemistry (n = 105). The expression of p-STAT3 or c-Myc was significantly higher in PCs with high SHCBP1 expression (Figure 9A, 9B), whereas tumors with low SHCBP1 expression were mostly low for p-STAT3 or c-Myc staining (Figure 9A, 9B). Additionally, GSEA analysis on the GSE57955 dataset confirmed that PC cases with high SHCBP1 expression (n = 19) also exhibited JAK-STAT3 signaling pathway activation, as compared with those with low high SHCBP1 expression (n = 20) (Figure 9C). Nevertheless, the correlated expression between SHCBP1 and c-Myc could also be seen in the GSE57955 dataset (Figure 9D). Therefore, these data suggested that the SHCBP1 function might be required for the STAT3/c-Myc signaling activation in PC.

Figure 9.

Figure 9

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A. Representing micrographs showed consistent expression of SHCBP1, p-STAT3 and c-Myc in SHCBP1-high or SHCBP1-low PC tissues. Bars: 100 μm. B. SHCBP1 expression was correlated with p-STAT3 or c-Myc in PC samples (n = 105). C. GSEA analysis revealed that SHCBP1-high PC cases exhibited highly enriched JAK-STAT3 signaling in GSE57955 dataset (n = 39). D. The mRNA expression of SHCBP1 is significantly correlated with c-Myc expression in GSE57955 (n = 39).

Discussion

PC is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. A better understanding of the molecular alterations involved in PC progression would lead to the identification of molecular markers, providing new targets for therapeutics. Recently, SHCBP1 was closely associated with cancer development in that aberrant expression of SHCBP1 has been documented in gastric, breast, and lung cancer [19,21,24]. These findings would implicate SHCBP1 as a potential oncogene in cancer development.

This study found that SHCBP1 expression was increased in PC compared with that in normal tissues. Although HPV is a significant contributing factor for approximately 30%-40% of PC, our findings did not reveal the association between SHCBP1 expression and HPV status, suggesting that the aberrant SHCBP1 expression in PC might not be caused by HPV infection. Moreover, we showed that SHCBP1 expression was significantly associated with clinicopathological parameters, including pathological grade, T stage, nodal status, and pelvic LNM, and could serve as an independent prognostic factor for overall survival in PC. Cellular functional analysis showed that the depletion of SHCBP1 expression suppressed cell proliferation, clonogenesis, and migration/invasion, and induced apoptosis in PC cells. Therefore, these findings suggest that SHCBP1 serves as a potential cancer marker for PC and exerts important oncogenic function during PC progression.

The function of SHCBP1 has been proposed to be related to cancer-related signaling pathways, including NF-κB, TGF-β/Smad, and β-catenin. In glioma, SHCBP1 promotes cell migration and invasion by activating the NF-κB signaling pathway [22]; SHCBP1 could also promote the metastasis of synovial sarcoma by regulating the TGF-β/Smad signaling pathway [20]. Recently, Liu et al. showed that SHCBP1 could activate EGF-induced β-Catenin signaling and promote cancer progression in lung cancer cells [34]. Therefore, SHCBP1 might serve as a crucial mediator that integrates growth factor signaling (TGF-β, EGF) to modulate tumor progression of cancer cells. As EGFR is frequently overexpressed in PC and may have an important role in PC carcinogenesis [35-37], it is reasonable to hypothesize that SHCBP1 might interact with key components of EGFR signaling and regulate malignant phenotypes of PC cells.

STAT3 is an essential signaling protein engaged by EGFR to promote cellular growth, differentiation, and survival in cancer cells [33,38]. Recently, STAT3 was identified as one of the top altered therapeutic targets in PC [39]. In this study, we identified STAT3/c-Myc signaling as a potential SHCBP1 downstream target. The SHCBP1:JAK2: STAT3 interaction positively regulates STAT3 activation and impacts on c-Myc expression in PC cells. Furthermore, disruption of STAT3/c-Myc signaling attenuated cell proliferation and cell migration/invasion in PC cells, while overexpression of constitutively activated STAT3 (STAT3CA) or c-Myc rescued cell proliferation and cell migration/invasion caused by SHCBP1 depletion in penile cancer cells. Therefore, the STAT3/c-Myc pathway might be a crucial downstream signaling of SHCBP1 in PC. In addition, the difference in downstream signaling activation regulated by SHCBP1 in PC and other cancer types might reflect the differential requirement of cancer cells to maintain their tumorigenic potential.

The finding that SHCBP1 regulates cell proliferation and tumorigenesis of PC cells sparked our interest in testing the effect of SHCBP1 depletion on PC cells in vivo. The effect of SHCBP1 depletion was confirmed by an in vivo xenograft study in which the transplanted tumors in the SHCBP1 knockdown group grew slower than those in the Scr control group. Consistent with the in vitro findings, SHCBP1 depletion attenuated STAT3/c-Myc signaling and induced apoptosis in the xenograft model. More important, correlated expression of SHCBP1, p-STAT3, and c-Myc was observed in penile cancer tissues, confirming the clinical relevance of SHCBP1/STAT3/c-Myc signaling in PC. In conclusion, aberrant SHCBP1 expression might serve as a potential prognostic biomarker for PC. SHCBP1 might be a potential oncogene in regulating STAT3/c-Myc signaling activation and tumor progression in vitro and in vivo in PC. Our findings highlight the potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target for PC, which may warrant further investigation in the future.

Acknowledgements

This work was supported by National Nature Science Foundation of China (Grant No. 81902605); Natural Science Foundation of Hunan Province, China (Grant No. 2020JJ5899, 2020JJ5915).

Disclosure of conflict of interest

None.

Supporting Information

ajcr0010-3138-f10.pdf (550.5KB, pdf)

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