Abstract
CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345, pCHK1Ser317, pCHK1Ser296, and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization‐dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.
Highlights
Vulvar carcinoma is a rare genital cancer counting for 3-5 % of all gynecological carcinomas and with an incidence ranging from 1 to 2 per 100 000 person-years worldwide.[1,2] In Norway, the overall incidence of vulvar squamous cell carcinomas (VSCC) has increased from 1.70 to 4.66 per 100 000 person-years in past 50 years.[3]
In 10 cases of normal vulvar squamous epithelium, no cytoplasmic immunoreactivity was observed for pCHK1Ser[345] and pCHK1Ser[296], whereas cytoplasmic staining for pCHK1Ser[317] and pCHK1Ser[280] was detected in basal, parabasal, middle, and top layers
Nuclear immunostaining was not seen for pCHK1Ser[345], and for pCHK1Ser[296], nuclear immunoreactivity was identified in basal and parabasal layer, whereas for pCHK1Ser[317] and pCHK1Ser[280], nuclear staining was detected in basal, parabasal, middle, and top layers (Figure 1A-D)
Summary
Vulvar carcinoma is a rare genital cancer counting for 3-5 % of all gynecological carcinomas and with an incidence ranging from 1 to 2 per 100 000 person-years worldwide.[1,2] In Norway, the overall incidence of VSCC has increased from 1.70 to 4.66 per 100 000 person-years in past 50 years.[3]. Alteration of expression and activation of CHK1 has been identified in a variety of cancer types, including glioblastoma,[14] sarcoma,[15] breast,[16] and colorectal cancer.[17] In breast cancer, abnormal expression of CHK1 correlated with advanced tumor behavior.[16] only few reports have studied activation and phosphorylation status of CHK1 with main focus on pCHK1Ser345.15,17. Given the crucial role CHK1 plays at cell cycle checkpoint and the fact that cancer cells rely mainly on ATR/ CHK1/CDC25C pathway to maintain genomic integrity due to dysfunctional ATM/CHK2/p53 pathway, therapeutic targeting of CHK1 has been investigated in the clinical trials both as a single agent and in combination with chemotherapy or radiotherapy.[18] In some cancer types, preferential killing of p53-deficient cells has been reported following CHK1 inhibition.[19] To our knowledge, neither the activation status nor the therapeutic potential of CHK1 targeting has been studied in vulvar cancer previously. As p53 mutations have previously been reported in 44%20 to 90%21 of vulvar cancers, we evaluated effects of the CHK1 targeting in p53 mutant vulvar cancer cell lines in vitro
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