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Importins and exportins represent an integral part of the nucleocytoplasmic transport machinery with fundamental importance for eukaryotic cell function. A variety of malignancies including hepatocellular carcinoma (HCC) show de-regulation of nuclear transport factors such as overexpression of the exportin Cellular Apoptosis Susceptibility (CAS). The functional implications of CAS in hepatocarcinogenesis remain, however, poorly understood. Here we integrated proteomics, transcriptomics and functional assays with patient data to further characterize the role of CAS in HCC. By analyzing ~ 1700 proteins using quantitative mass spectrometry in HCC cells we found that CAS depletion by RNAi leads to de-regulation of integrins, particularly down-regulation of integrin β1. Consistent with this finding, CAS knockdown resulted in substantially reduced migration and invasion of HCC cell lines as analyzed by 2D 'scratch' and invasion chamber assays, respectively. Supporting the potential in vivo relevance, high expression levels of CAS in HCC tissue samples were associated with macroangioinvasion and poorer patient outcome. Our data suggest a previously unanticipated link between CAS and integrin signaling which correlates with an aggressive HCC phenotype., Competing Interests: None.

Thyroid carcinoma is among the most common malignant endocrine neoplasms with a rising incidence. Genetic alterations occurring in thyroid cancer frequently affect the RAS/RAF/MEK/ERK-pathway such as the oncogenic, kinase-activating BRAF(V600E) mutation. Nuclear transport receptors including importins and exportins represent an important part of the nuclear transport machinery providing nucleo-cytoplasmic exchange of macromolecules. The role of nuclear transport receptors in the development and progression of thyroid carcinomas is largely unknown. Here, we studied the expression and function of the exportin cellular apoptosis susceptibility (CAS) in thyroid carcinogenesis and its link to the BRAF(V600E) mutation. By using immunohistochemistry (IHC) we found significantly increased IHC scores of CAS in primary papillary (PTC) and medullary (MTC), but not in follicular (FTC) thyroid carcinoma compared to non-tumorous (NT) thyroid tissue. Interestingly, metastases of the aforementioned subtypes including FTC showed a strong CAS positivity. Among PTCs we observed that CAS immunoreactivity was significantly higher in the tumors harboring the BRAF(V600E) mutation. Furthermore, depletion of CAS by RNAi in the BRAF(V600E)-positive PTC cell line B-CPAP led to reduced tumor cell growth measured by crystal violet assays. This phenotype could be attributed to reduced proliferation and increased cell death as assayed by BrdU ELISAs and immunoblotting for PARP-cleavage, respectively. Finally, we found additive effects of CAS siRNA and vemurafenib treatment in B-CPAP cells. Collectively, these data suggest that CAS overexpression in thyroid carcinoma depends on the subtype and the disease stage. Our findings also indicate that CAS maintains PTC cell proliferation and survival. Targeting CAS could represent a potential therapeutic approach particularly in combination with BRAF inhibitors such as vemurafenib in BRAF(V600E)-positive tumors.

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., (© 2014 by the American Association for the Study of Liver Diseases.)