Aberrant CEACAM19 expression is associated with metastatic phenotype in penile cancer

Abstract

Objective: A greater knowledge of the mechanisms of the pathogenesis of penile cancers may assist in the development of more tailored targeted therapy. Herein, we aimed to evaluate the expression of CEACAM19 in penile cancer and to explore its regulatory mechanisms.

Material and methods: This retrospective study enrolled 64 penile cancer patients who underwent penectomy between 2011 and 2015. CEACAM19 expression in tissues was detected by immunohistochemistry, which was analyzed in association with clinicopathological parameters. Kaplan-Meier analysis was performed to evaluate the relationship between CEACAM19 expression and prognosis of patients with penile cancer. Cell Counting Kit-8 assay and clonogenic assay were used to evaluate the cell viability and tumorigenic potential of penile cancer cell line, respectively; wound healing assay and transwell invasion assay were conducted to evaluate the effect of CEACAM19 depletion on cell migration and invasion in penile cancer cells; CEACAM19 protein expression was analyzed by Western blotting. Culture supranatant matrix metalloproteinase 2/9 (MMP2/9) was detected by ELISA.

Results: CEACAM19 was differentially expressed in non-cancerous and penile cancer tissues. Over-expression of CEACAM19 was significantly associated with nodal and distant metastasis, and predicted unfavorable cancer-specific survival in penile cancer. Depletion of CEACAM19 expression suppressed cell proliferation, reduced colony formation, and attenuated cell migration and invasion in Penl1 cells. Furthermore, knockdown of CEACAM19 expression attenuated the levels of p-Smad2/3 and reduced secretion of MMP2/9 in Penl1 cells. The effects of CEACAM19 might result from its function in regulating the Smad2/3 activation, as inhibition on Smad2/3 activation suppressed cell migration and invasion and reduced MMP2/9 secretion in Penl1 cells.

Conclusion: Over-expression of CEACAM19 might serve as a potential prognostic biomarker for clinical management of penile cancer. Strategies targeting CEACAM19-regulated signaling pathways may have a therapeutic benefit in penile cancer.

Keywords: CEACAM19; metastasis; penile cancer; prognosis.

Figure 1

Notes: (A) Expression of CEACAM19 in non-cancer penile tissues and penile cancer. Bars: 100 µm. Upper panel, low expression of CEACAM19 in non-cancer penile tissues (1–3); middle panel, penile cancer cases with over-expression of CEACAM19 (Case 1–3); lower panel, penile cancer cases with low expression of CEACAM19 (Case 4–6). (B) Over-expression of CEACAM19 in penile cancer was associated with unfavorable CSS. (C) Nodal status predicted unfavorable CSS in penile cancer. The log-rank test was used to compare survival curves. Abbreviation: CSS, cancer-specific survival.

Figure 2

Knockdown of CEACAM19 expression suppresses cell growth and clonogenesis in Penl1 cells. Notes: (A) CEACAM19 expression was significantly reduced in Penl1 cells transduced with shCEACAM19 lentivirus. β-Actin served as a loading control. (B) Knockdown of CEACAM19 expression attenuated cell growth of Penl cells. The CCK-8 absorbance was measured at 450 nm (OD₄₅₀). *P<0.05, Scr vs shCEACAM19. (C) Depletion of CEACAM19 expression reduced clonogenesis of Penl1 cells. The colony formed in Scr control was regarded as 100%. *P<0.05, Scr vs shCEACAM19. All experiments were performed three times, and data are presented as mean ± SD. values. *P<0.05, Scr vs shCEACAM19. Abbreviations: CCK-8, Cell Counting Kit-8; Scr, scramble; shCEACAM19, shRNA targeting CEACAM19.

Figure 3

Depletion of CEACAM19 expression inhibits cell migration and invasion in Penl1 cells. Notes: (A) Wound healing assay. The migration of the cells to the wound was measured at 0 and 24 hours after scratch. The representative fields were photographed; the relative healing rates were quantified with measurements of the gap sizes after the culture. Three different areas in each assay were chosen to measure the distances of migrating cells to the origin of the wound. Bars: 100 µm. *P<0.05, Scr vs shCEACAM19. (B) Transwell invasion assays with Penl1 cells were performed in Scr control and shCEACAM19 group. Crystal violet assay (OD₅₇₀) was conducted to evaluate cell migration and invasion capability. Bars: 100 µm. All experiments were performed three times, and data are presented as mean ± SD. values. *P<0.05, Scr vs shCEACAM19. Abbreviations: Scr, scramble; shCEACAM19, shRNA targeting CEACAM19.

Figure 4

Notes: (A) Multiple pathway reporter analysis identified potential cancer-related pathways regulated by CEACAM19 in Penl1 cells. (B) Knockdown of CEACAM19 expression attenuated p-Smad2/3 expression in Penl1 cells. β-Actin served as a loading control. (C) Knockdown of CEACAM19 expression reduced MMP2/9 secretion in Penl1 cells. All experiments were performed three times, and data are presented as mean ± SD values. *P<0.05, Scr vs sh CEACAM19.

Figure 5

Notes: (A) The effect of SB431542 (SB) on p-Smad2/3 levels in Penl1 cells. Penl1 cells were treated with 10 µM SB for 24 hours. β-Actin served as a loading control. (B) The effect of SB431542 on wound healing of Penl1 cells. Three different areas in each assay were chosen to measure the distances of migrating cells to the origin of the wound. *P<0.05, Control vs SB. (C) The effect of SB431542 on cell invasion of Penl1 cells. Transwell invasion assay was conducted to evaluate cell migration and invasion capability. *P<0.05, Control vs SB. (D) Inhibition on TGF-β/Smad2/3 activity by SB431542 reduced MMP2/9 secretion in Penl1 cells. All experiments were performed three times, and data are presented as mean ± SD. values. *P<0.05, Control vs SB.