Aberrant expression of PDZ-binding kinase/T-LAK cell-originated protein kinase modulates the invasive ability of human pancreatic cancer cells via the stabilization of oncoprotein c-MYC.

High frequency of mortality in patients with pancreatic ductal adenocarcinoma (PDAC) is vastly associated with the invasive and metastatic nature of these cancer cells. Little is known about the factors involved in this invasive/metastatic process. The current challenge in the treatment of these patients is the lack of viable options besides gemcitabine. The aim of this study was to evaluate the role of PDZ-binding kinase (PBK)/T-LAK cell-originated protein kinase (TOPK) in invasive PDAC cells and to determine whether PBK/TOPK expression drives invasiveness in PDAC. Using gain-of-function and loss-of-function studies in established and patient-derived xenograft-PDAC cell lines, and examining patient-derived archival tissue samples, we demonstrate for the first time that PBK/TOPK is upregulated in pancreatic cancer and expression levels are closely associated with the invasive property of pancreatic cancer cells. Modulation of PBK/TOPK causally regulates the invasive ability of PDAC cells. We also demonstrate that two key players in metastatic invasion, matrix metalloproteinases-2 (MMP-2) and MMP-9 gelatinase activity and gene promoter activities, are regulated by PBK/TOPK. Moreover, we demonstrate for the first time that PBK/TOPK provides stability of an oncoprotein, c-MYC, which transcriptionally regulates MMP-2 and MMP-9 in these invasive PDAC cells. Our in vitro and in situ data corroborate that PBK/TOPK is closely associated with the invasive nature of PDAC and reveal a novel mechanism by which the metastatic behavior of human pancreatic cancer cells is regulated. These findings provide a rationale for targeting PBK/TOPK for the therapeutic intervention of invasive/metastatic pancreatic cancer in human.

CRISPR-Cas9 Knockdown and Induced Expression of CD133 Reveal Essential Roles in Melanoma Invasion and Metastasis.

CD133, known as prominin1, is a penta-span transmembrane glycoprotein presumably a cancer stem cell marker for carcinomas, glioblastomas, and melanomas. We showed that CD133(+) 'melanoma-initiating cells' are associated with chemoresistance, contributing to poor patient outcome. The current study investigates the role(s) of CD133 in invasion and metastasis. Magnetic-activated cell sorting of a melanoma cell line (BAKP) followed by transwell invasion assays revealed that CD133(+) cells are significantly more invasive than CD133(-) cells. Conditional reprogramming of BAKP CD133(+) cells maintained stable CD133 overexpression (BAK-R), and induced cancer stem cell markers, melanosphere formation, and chemoresistance to kinase inhibitors. BAK-R cells showed upregulated CD133 expression, and consequently were more invasive and metastatic than BAK-P cells in transwell and zebrafish assays. CD133 knockdown by siRNA or CRISPR-Cas9 (BAK-R-T3) in BAK-R cells reduced invasion and levels of matrix metalloproteinases MMP2/MMP9. BAK-R-SC cells, but not BAK-R-T3, were metastatic in zebrafish. While CD133 knockdown by siRNA or CRISPR-Cas9 in BAK-P cells attenuated invasion and diminished MMP2/MMP9 levels, doxycycline-induced CD133 expression in BAK-P cells enhanced invasion and MMP2/MMP9 concentrations. CD133 may therefore play an essential role in invasion and metastasis via upregulation of MMP2/MMP9, leading to tumor progression, and represents an attractive target for intervention in melanoma.

CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition.

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibition in vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as "melanoma initiating cells," resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

Targeting the Root Cause of Cystic Fibrosis.

Cystic Fibrosis (CF) is a serious genetic condition caused by CF transmembrane conductance regulator (CFTR) mutation. CF patients have shortened lifespan due to airway obstruction, infection, and end-stage lung failure. However, recent development in CF therapy suggests a brighter future for CF patients. Targeting specific CFTR mutations aims to potentiate the channel gating activity of impaired CFTR and restore protein trafficking to the plasma membrane. Gene therapy introduces correct CFTR gene into the affected airway epithelium leading to the functional expression of CFTR in CF patients. This review will sum up the current status in CF-cause targeting therapy.