Abstract
Oncogenic KRAS mutation plays a key role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis with nearly 95% of PDAC harboring mutation-activated KRAS, which has been considered an undruggable target. Doublecortin-like kinase 1 (DCLK1) is often overexpressed in pancreatic cancer, and recent studies indicate that DCLK1+ PDAC cells can initiate pancreatic tumorigenesis. In this study, we investigate whether overexpressing DCLK1 activates RAS and promotes tumorigenesis, metastasis, and drug resistance. Human pancreatic cancer cells (AsPC-1 and MiaPaCa-2) were infected with lentivirus and selected to create stable DCLK1 isoform 2 (alpha-long, AL) overexpressing lines. The invasive potential of these cells relative to vector control was compared using Matrigel coated transwell assay. KRAS activation and interaction were determined by a pull-down assay and coimmunoprecipitation. Gemcitabine, mTOR (Everolimus), PI3K (LY-294002), and BCL-2 (ABT-199) inhibitors were used to evaluate drug resistance downstream of KRAS activation. Immunostaining of a PDAC tissue microarray was performed to detect DCLK1 alpha- and beta-long expression. Analysis of gene expression in human PDAC was performed using the TCGA PAAD dataset. The effects of targeting DCLK1 were studied using xenograft and Pdx1CreKrasG12DTrp53R172H/+ (KPC) mouse models. Overexpression of DCLK1-AL drives a more than 2-fold increase in invasion and drug resistance and increased the activation of KRAS. Evidence from TCGA PAAD demonstrated that human PDACs expressing high levels of DCLK1 correlate with activated PI3K/AKT/MTOR-pathway signaling suggesting greater KRAS activity. High DCLK1 expression in normal adjacent tissue of PDAC correlated with poor survival and anti-DCLK1 mAb inhibited pancreatic tumor growth in vivo in mouse models.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of any major malignancy with less than an 8%5-year survival rate and is the third leading cause of cancerrelated deaths in the United States [1]
Doublecortin-like kinase 1 (DCLK1) is often overexpressed in pancreatic cancer and is coexpressed with other PDAC cancer stem cell-like (CSC) markers, and recent studies indicate that DCLK1+ PDAC cells can initiate pancreatic tumorigenesis in the presence of mutation and inflammation [8, 9]
We have demonstrated that DCLK1 regulates key oncogenes, pluripotency factors, angiogenic factors, epithelial mesenchymal transition (EMT) related transcription factors, and pancreatic cancer xenograft growth which can be reversed by downregulating DCLK1 or inhibiting its kinase activity [10,11,12,13]
Summary
Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of any major malignancy with less than an 8%5-year survival rate and is the third leading cause of cancerrelated deaths in the United States [1]. Active KRAS directs several downstream signaling pathways that play pivotal roles in proliferation, migration, invasion, and survival, which are the most important cellular mechanisms regulating PDAC tumorigenesis and metastasis. Cells with cancer stem cell-like (CSC) properties have been identified in PDAC. These cells are often resistant to conventional chemotherapy and radiation therapy and as such may explain why current treatments do not cure PDAC or prevent recurrences. Doublecortin-like kinase 1 (DCLK1) is often overexpressed in pancreatic cancer and is coexpressed with other PDAC CSC markers, and recent studies indicate that DCLK1+ PDAC cells can initiate pancreatic tumorigenesis in the presence of mutation and inflammation [8, 9]. We have demonstrated that DCLK1 regulates key oncogenes, pluripotency factors, angiogenic factors, epithelial mesenchymal transition (EMT) related transcription factors, and pancreatic cancer xenograft growth which can be reversed by downregulating DCLK1 or inhibiting its kinase activity [10,11,12,13]
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