Correlation of CD44v6 expression with ovarian cancer progression and recurrence

Abstract

Background: Previously some groups demonstrated that CD44 variant 6 (CD44v6) is correlated with progression and metastasis of ovarian cancer. However, a number of other groups failed to find such an association. Moreover, epithelial ovarian cancer is known to easily metastasize to distinct sites such as the pelvic and abdominal cavities, but the potential association of CD44v6 expression with site-specific metastasis of ovarian cancer has not been explored. This study sought to evaluate the expression of CD44 standard (CD44s) and CD44v6 in primary, metastatic and recurrent epithelial ovarian cancer to explore the potential association of CD44s and CD44v6 with tumor progression and recurrence.

Methods: Tumor specimens were procured from patients with advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma. CD44s and CD44v6 expression in the tumor tissues was evaluated by real-time RT-PCR and Western blot. Moreover, serum soluble CD44s or CD44v6 concentrations of early stage (FIGO I, G1), advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma patients were determined by enzyme-linked immunosorbent assays (ELISA). CD44v6 expression in a different set of tumor samples on an ovarian cancer tissue chip was evaluated by immunohistochemistry (IHC) and the correlation of CD44v6 expression with clinicopathologic features was analyzed. Finally, the effects of knockdown of CD44v6 in SKOV3 cells on cell adhesion, invasion and migration were assessed.

Results: The expression of CD44v6, but not CD44s, is up-regulated in recurrent ovarian serous cancer compared to advanced primary tumor. CD44v6 expression is also preferentially increased in the tumor at the abdominal cavity metastasis site of advanced diseases. Consistently, serum soluble CD44v6 levels of recurrent ovarian cancer were higher than those of early stage and advanced primary diseases. The IHC data demonstrate that CD44v6 expression is correlated with clinicopathologic features and tumor progression. Lastly, knockdown of CD44v6 decreases the adhesion and migration but not invasion capacities of SKOV3 cells.

Conclusions: CD44v6 expression levels are associated with epithelial ovarian cancer progression, metastasis and relapse. Moreover, serum soluble CD44v6 may be used as a potential marker for identifying tumor relapse. Finally, CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration.

Figure 1

Expression of CD44 and CD44v6 in advanced primary and recurrent epithelial ovarian cancer tissues, and serum concentrations of soluble CD44s (sCD44s) and CD44v6 (sCD44v6) in patients with early-stage, advanced-stage and recurrent epithelial ovarian cancer. (A) Real-time RT-PCR analysis of mRNA levels of CD44 (using a pair of primers complementary to common regions of CD44s and CD44v) and CD44v6 (using a pair of primers specific to CD44v6) in 45 advanced primary tumor samples and 20 tissues from recurrent diseases. **: P<0.01. (B) Western blot analysis of CD44s and CD44v6 protein levels in the same set of advanced primary and recurrent tumor samples as in A. *: P<0.05. (C) Representative images of Western blots performed in B. (D) Serum levels of sCD44s and sCD44v6 in peripheral blood samples from patients with early-stage, advanced-stage and recurrent epithelial ovarian cancer were determined by ELISA as described in Methods. The ELISA was repeated three times. **: P<0.01.

Figure 2

Expression of CD44 and CD44v6 in tumor samples from different locations in the same patients. (A) Real-time RT-PCR analysis of mRNA levels of CD44 (using a pair of primers complementary to common regions of CD44s and CD44v) and CD44v6 (using a pair of primers specific to CD44v6) in tumor tissues from the primary site, the abdominal cavity metastasis (Met-1) and the pelvic cavity metastasis (Met-2) of 28 patients with advanced ovarian cancer. **: P<0.01. (B) Western blot analysis of CD44s and CD44v6 protein levels in the same set of tumor samples as in A. *: P<0.05. (C) Representative images of Western blots performed in B.

Figure 3

IHC analysis of CD44v6 expression in tumor tissues on an Ovarian Cancer Tissue Chip. Representative IHC staining images of grade 3 ovarian serous adenocarcinoma (A) and corresponding negative control (D), grade 2 lymph node metastatic serous adenocarcinoma (B) and corresponding negative control (E), and edge tissues of normal ovary (C) and corresponding negative control (F).

Figure 4

Efficient knockdown of CD44v6 in SKOV3 cells by transfected siRNA against CD44v6. (A) Quantitative real-time RT-PCR analysis of mRNA levels of CD44v6 expression in parental SKOV3 cells and SKOV3 cells transiently transfected with control RNA, siRNA-NC, siRNA-1 and siRNA-2. **: P<0.01. (B) Western blot analysis of CD44v6 protein levels in the same set of SKOV3 cell as in A. *: P<0.05. (C) Representative images of Western blots performed in B.

Figure 5

Assessment of the adhesion, invasion and migration capacities of SKOV3 cells with CD44v6 knockdown. (A) Cell adhesion assays with parental SKOV3 cells and SKOV3 cells transiently transfected with siRNA-NC, siRNA-1 and siRNA-2. (B) Cell invasion assays with parental SKOV3 cells (a) and SKOV3 cells transiently transfected with siRNA-NC (b), siRNA-1 (c) and siRNA-2 (d). (C) Cell migration assays with parental SKOV3 cells (a) and SKOV3 cells transiently transfected with siRNA-NC (b), siRNA-1 (c) and siRNA-2 (d).