Prosurvival function of the cellular apoptosis susceptibility/importin-α1 transport cycle is repressed by p53 in liver cancer.

UNLABELLED: Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-α1 (imp-α1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-α1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-α1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-α1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner. CONCLUSION: The CAS/imp-α1 transport cycle is linked to XIAP and is required to maintain tumor cell survival in HCC. Moreover, CAS and imp-α1 are targets of p53-mediated repression, which represents a novel aspect of p53's ability to control tumor cell growth in hepatocarcinogenesis.

Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells.

Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of α-tubulin with ß-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with α-tubulin and ß-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of α-tubulin and ß-tubulin, increased α-tubulin and ß-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes.

The p53 tumor suppressor protein regulates many genes that can determine different cellular outcomes such as growth arrest or cell death. Promoter-selective transactivation by p53, although critical for the different cellular outcomes, is not well understood. We report here that the human cellular apoptosis susceptibility protein (hCAS/CSE1L) associates with a subset of p53 target promoters, including PIG3, in a p53-autonomous manner. Downregulation of hCAS/CSE1L decreases transcription from those p53 target promoters to which it preferentially binds and reduces apoptosis. In addition, hCAS/CSE1L silencing leads to increased methylation of histone H3 lysine 27 within the PIG3 gene. hCAS/CSE1L was previously shown to function as a nucleo-cytoplasmic transport factor, as does its closely related yeast homologue Cse1, which can also associate with chromatin and serve as a barrier protein that prevents spreading of heterochromatin. Thus, human CAS/CSE1L can bind select genes with significant functional consequences for p53-mediated transcription and determine cellular outcome.

Src and Cas are essentially but differentially involved in angiotensin II-stimulated migration of vascular smooth muscle cells via extracellular signal-regulated kinase 1/2 and c-Jun NH2-terminal kinase activation.

Angiotensin II (Ang II) plays an important role in several cardiovascular diseases associated with vascular smooth muscle cell (VSMC) growth and migration. Src activity is known to be required for the migration of a number of cell types. p130Cas was reported to be essential for cell migration and actin filament reorganization. Mitogen-activated protein (MAP) kinases were also reported to be critical regulatory factors for growth and migration of VSMC. However, precise intracellular mechanisms involving c-Src, p130Cas, and MAP kinases in Ang II-stimulated migration of VSMC have not been well elucidated. Here we demonstrated that Ang II rapidly and significantly stimulated tyrosine phosphorylation of Src and Cas and their association in rat aortic smooth muscle cells (RASMC). Ang II-stimulated tyrosine phosphorylation of Src and Cas and activation of ERK1/2 and JNK, but not p38, were potently inhibited by Src family tyrosine kinase inhibitors, herbimycin A (HA) and PP2. Ang II-stimulated Src and Cas association, tyrosine phosphorylation of Cas, and activation of ERK1/2 and JNK were suppressed in kinase-inactive Src (KI Src)-overexpressed RASMC. Ang II-stimulated JNK activation but not ERK1/2 activation was blocked in substrate domain-deleted Cas (DeltaSD Cas)-overexpressed RASMC. In addition, HA, PP2, ERK1/2 inhibitor, 2'-amino-3'-methoxyflavone (PD98059) and JNK inhibitor, and anthra[1,9-cd]pyrazol-6(2H)-one (SP600125) significantly inhibited Ang II-stimulated migration of RASMC. Ang II-induced colocalization of Src and Cas and migration were inhibited in both KI Src- and DeltaSD Cas-overexpressed RASMC. These findings suggest that Src and Cas are essentially but differentially involved in Ang II-stimulated migration of VSMC through the activation of ERK1/2 and JNK.

CSE1L/CAS: its role in proliferation and apoptosis.

CAS/CSE1L is the human homologue of the yeast gene CSE1. It was first cloned while searching for genes that rendered breast cancer cells resistant towards toxin induced apoptosis. Since depletion of CSE1 leads to cell-cycle arrest, CAS is thought to be involved in proliferation. CAS functions in the mitotic spindle checkpoint. CAS is located on chromosome 20q13, a locus often amplified in cancers of various origin, e.g. colonic or breast cancer. Since genetic instability is a hallmark of cancer, amplification or over expression of the CAS gene might interfere with or override its role in the mitotic spindle checkpoint. CAS is also implicated in the nuclear to cytoplasmic reshuffling of importin alpha, which itself is necessary for the nuclear transport of several proliferation activating proteins, transcription factors, oncogene and tumor suppressor gene products such as p53 and BRCA1. Inhibition of MEK1 mediated phosphorylation has been shown to enhance paclitaxel (Taxol) induced apoptosis in breast, ovarian, and lung tumor cell lines in-vitro. Since CAS is also phosphorylated (activated) by MEK1, and since the anti-cancer drug Taxol alters the microtubule assembly and activates pro-apoptotic signaling pathways, altering the activity/phosphorylation status of CAS via MEK1 inhibition may present a potential strategy in experimental cancer therapy.