Association of epithelial-mesenchymal transition with the metastatic potential of disseminated cells in breast cancer was studied.

We have identified CALNUC, an EF-hand, Ca2+-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gαi3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gαi3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca2+-binding protein detected by 45Ca2+-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.

We previously demonstrated that CALNUC, a Ca²⁺-binding protein with two EF-hands, is the major Ca²⁺-binding protein in the Golgi by ⁴⁵Ca²⁺ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515–1527). In this study we investigated CALNUC9s properties and the Golgi Ca²⁺ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 μg CALNUC/mg Golgi protein, 2.5 × 10⁵ CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca²⁺-binding site (K_d = 6.6 μM, binding capacity = 1.1 μmol Ca²⁺/μmol CALNUC). ⁴⁵Ca²⁺ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand α helix from CALNUC completely abolished its Ca²⁺-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, α-mannosidase II (Man II). Approximately 70% of the ⁴⁵Ca²⁺ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca²⁺ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP₃ alone (permeabilized cells) also resulted in a significant increase in Ca²⁺ release from Golgi stores. By immunofluorescence, the IP₃ receptor type 1 (IP₃R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca²⁺ in vivo and together with SERCA and IP₃R is involved in establishment of the agonist-mobilizable Golgi Ca²⁺ store.

γ-Secretase, which is responsible for the intramembranous cleavage of Alzheimer β-amyloid precursor protein and the signaling receptor Notch, is a multiprotein complex consisting of at least four components: presenilin (PS); nicastrin (Nct); APH-1 (anterior pharynx-defective-1); and presenilin enhancer-2 (PEN-2). Presenilin 1 (PS1) is known to be essential for the stability, interaction, and trafficking of the other PS1/γ-secretase components. However, the precise functions of the other components remain elusive. Here, we investigated the functions of Nct within the PS1/γ-secretase complex. We demonstrated that the loss of Nct expression in the embryonic fibroblast cells (Nct KO cells) results in dramatically decreased levels of APH-1, PEN-2, and PS1 fragments accompanied by a significant accumulation of full-length PS1. In the absence of Nct, PEN-2 and full-length PS1 are subjected to proteasome-mediated degradation, whereas the degradation of APH-1 is mediated by both proteasomal and lysosomal pathways. Unlike the case of wild type cells in which the γ-secretase complex mainly locates in the trans-Golgi network, the majority of residual PEN-2, APH-1, and the uncleaved full-length PS1 in Nct KO cells reside in the endoplasmic reticulum, which remain associated with each other in the absence of Nct. Interestingly, significant amounts of full-length PS1 and PEN-2, but not APH-1, are detected on the plasma membrane in Nct KO cells, suggesting the Nct-independent cell surface delivery of the PEN-2·PS1. Finally, the diminished PEN-2 protein level in Nct-deficient cells can be partially restored by overexpression of exogenous PS1, APH-1, or PEN-2 individually or collectively, indicating a dispensable role for Nct in controlling PEN-2 level. Taken together, our study demonstrates a critical role of Nct in the stability and proper intracellular trafficking of other components of the PS1/ γ-secretase complex but not in maintaining the association of PEN-2, APH-1, and full-length PS1.

Lung cancer is the leading cancer of all cancer-related death in most countries. Roughly 20% are from small cell lung cancer (SCLC), and 80% are non-small cell lung cancer (NSCLC) which includes adenocarcinoma (ADC) and squamous cell carcinoma (SCC).¹ x 1 Sher, YP, Shih, JY, Yang, PC et al. Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with multiple marker genes. Clin Cancer Res. 2005; 11: 173–179 PubMed | Google Scholar See all References Worldwide, 5-year survival rate of lung cancer patient is less than 5 to 15%² x 2 Jemal, A, Siegel, R, Ward, E et al. Cancer statistics, 2006. CA Cancer J Clin. 2006; 56: 106–130 Crossref | PubMed | Scopus (5099) | Google Scholar See all References Nearly 50% of early-stage lung cancer patients will relapse or develop metastases within 5 years after surgical removal of tumor mass, indicating the existence of occult metastatic cells which can not be effectively detected by current methods.

Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45-/CD31-) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM+ aneuploid circulating tumor stem cells (CTSCs), and EpCAM- small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence.