Although great efforts have been made in fighting pancreatic cancer in recent decades, the cancer still poses a serious threat to human life and health[1, 2]. The main reasons for the tragedy are that our understanding the mechanism of pancreatic cancer is shallow, and the effective drug against the cancer is unavailable in the clinical setting[3–6]. In the current study, we found that CD44 gene knockout effectively inhibited pancreatic cancer cell tumorigenesis, migration, and invasion mainly through down-regulating oncogenic Xist, suppressing tumorigenic AKT and ERK signaling pathways, and activating the tumor-suppressive p38-p53 signaling pathway. Our findings indicate that CRISPR/Cas9-mediated CD44 gene knockout is a new strategy for targeted pancreatic cancer therapy.
The tumor tissues of pancreatic cancer are rich in cancer stem cells which trigger tumor initiation, progression, metastasis, drug resistance, recurrence. Additionally, the numbers of cancer stem cells in the tumor tissues are closely associated with poor prognosis in pancreatic cancer patients[7–11]. Thus, cancer stem cells are a good target for cancer therapy. However, the approach that effectively removals cancer stem cells in malignant tumors is limited, and the therapy that targets pancreatic cancer stem cells is absent in the clinical setting[3]. In this study, we demonstrated in concept that CD44 gene knockout reduced pancreatic cancer cell stemness and tumorigenesis. Thereby, CD44 gene knockout is a new approach for eliminating pancreatic cancer stem cells and raising anti-pancreatic cancer efficacy.
In pancreatic cancer, overexpression of CD44, particularly in aberrant expression of various CD44 variants, is closely associated with poor prognosis in cancer patients[23–27]. However, effective CD44-targeted drug against pancreatic cancer is not available in the clinical setting. As mentioned before, the mRNA splicing error causes production of multiple CD44 variants; notably, many CD44 variants are constitutively activated in the tumor microenvironment, and these active CD44 variants escalate multiple downstream oncogenic genes and tumorigenic signaling pathways, therefore triggering pancreatic cancer initiation and progression[20–22]. Whereas, there is no effective therapy for CD44- and CD44 variants-targeted pancreatic cancer therapy at present, and CD44 gene knockout in pancreatic cancer has not been reported in the literature. In the current study, we found that CD44 gene knockout effectively inhibited pancreatic cancer cell tumorigenesis, migration, invasion, and tumor growth in xenograft mice. Our findings indicate that CD44 and CD44 variants are sensible targets for targeted pancreatic cancer therapy; thus, CD44 gene knockout catches all the gang of CD44s and numerous CD44 variants and is a new strategy for treatment of pancreatic cancer.
Xist is abnormally overexpressed in various types of malignant tumors, including pancreatic cancer[38–41]. In pancreatic cancer, Xist upregulates expression of oncogenic EGFR, YAP, ZEB1, TGF-β, Notch1, and Notch2, promotes pancreatic cancer cell proliferation, migration, and invasion, and triggers the initiation and development of many diseases, including cancer[42–49, 56]. However, the method that effectively inhibits expression of oncogenic Xist in cancer cells has not yet been reported in the literature. In this study, we found that CD44 gene knockout markedly suppressed Xist expression in pancreatic cancer cells, suggesting that CD44 controls Xist expression in cancer cells. Accordingly, we propose that there is a new oncogenic CD44-Xist signaling axis in cancer. To our knowledge, this is the first time for unraveling the novel CD44-Xist signaling axis in malignant tumor. Our findings provide a new approach for reducing oncogenic Xist expression in cancer cells, and offer a platform for developing novel therapy for inhibition of oncogenic CD44-Xist axis in malignant tumors.
The tumorigenic AKT and ERK are abnormally activated in pancreatic cancer, while tumor-suppressive p53 is usually deficient in malignant tumor. In the current study, we found that CD44 gene knockout not only suppressed tumorigenic AKT and ERK signaling pathways, but also concurrently activated the tumor-suppressive p38-p53 signaling pathway. Additionally, CD44 gene knockout induced DNA damage in pancreatic cancer cells and increased the sensibility of pancreatic cancer cells to the anti-cancer drug cisplatin, suggesting that CD44 gene knockout reduces drug resistance and increase cisplatin-mediated anti-pancreatic cancer efficacy.
CRISPR/Cas9 technology has recently been used in preclinical and primary clinical trials for treatment of diseases[51, 57, 58], and has shown promising therapeutic results[59, 60]. In this study, we found that CD44-sgRNA2 (CGCTACAGCATCTCTC GGA) and CRISPR-Cas9-mediated CD44 genomic disruption inhibited pancreatic cancer cell tumorigenesis. Our study demonstrated in concept that CD44 gene knockout is a potential approach for targeted pancreatic cancer therapy. In the future, CD44sgRNA2 and Cas9 can be packed in the HA-liposome nanoparticles as described in recent studies[51, 57, 58]. In this way, the HA-liposome nanoparticles-CD44sgRNA2 and Cas9 can bind to cancer stem/progenitor cells surface, and deliver CD44sgRNA2 and Cas9 into the cancer cells, consequently disrupting CD44 gene in vivo and inhibiting pancreatic cancer effectively. Our data suggest that CRISPR/Cas9-mediated CD44 gene knockout is a new approach for targeted treatment of pancreatic cancer.
In conclusion, CD44 gene knockout notably inhibited pancreatic cancer cell tumorigenesis, migration, invasion, increased intracellular DNA damage, sensitized pancreatic cancer cells to the anticancer drug cisplatin, and suppressed tumor growth in xenograft mice. CD44 gene knockout markedly reduced expression of oncogenic Xist, suppressed tumorigenic AKT and ERK signaling pathways, and concurrently activated the key tumor-suppressive p38 and p53 signaling pathways. Our findings highlight the critical role of CD44 in pancreatic cancer pathogenesis and provide a new strategy for targeted pancreatic cancer therapy.