Mengos, April

9082 Background: Human tumors, including melanoma, are complex mixtures of individual, molecularly distinct subpopulations, or clones of cancer cells. Effective cancer therapy will likely require targeting of all tumor subsets within a given cancer. Understanding the tumor complexity and the ability to identify points of therapeutic vulnerability within the individual tumor subsets will be essential for development of effective personalized cancer therapies. Methods: We have developed an approach that combines identification of individual tumor subsets using a multiparameter nuclear flow cytometry coupled with a high-resolution genomic analysis using the array-based comparative genomic hybridization (aCGH). Melanoma nuclei were isolated from tumor tissues and subjected to flow cytomery using melanocyte-specific antibodies (to separate melanoma cells from stroma) and DNA content, to separate individual tumor subpopulations. DNA extracted from isolated nuclear subpopulations was extracted and analyzed by aCGH. This approach was adopted for both fresh-frozen and paraffin-embedded clinical specimens. Results: We initially demonstrate the feasibility of the outlined approach by successful separation of melanoma from stromal nuclei and separation of individual melanoma nuclear subpopulations by DNA content. aCGH analysis of the DNA derived from isolated tumor subpopulations allowed successful identification of potentially targetable molecular aberrations in individual subsets of tumor cells. Notably, such aberrations were often not detected in unsorted, bulk tumors analyzed by the same high-resolution aCGH approach. Conclusions: We demonstrate a feasible approach to in-depth molecular analysis of tumor subpopulations within a clinical cancer tissue. This approach allows identification of potentially targetable molecular aberrations within individual tumor subsets, thus opening a possibility for a broad tumor targeting through design of individually-tailored therapeutic approaches. No significant financial relationships to disclose.

The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated ΔF508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.

Abstract Deletion of phenylalanine 508 (ΔF508) accounts for nearly 70% of all mutations that occur in the cystic fibrosis transmembrane conductance regulator (CFTR). The ΔF508 mutation is a class II processing mutation that results in very little or no mature CFTR protein reaching the apical membrane and thus no cAMP-mediated Cl− conductance. Therapeutic strategies have been developed to enhance processing of the defective ΔF508 CFTR molecule so that a functional cAMP-regulated Cl− channel targets to the apical membrane. Sarcoplasmic/endoplasmic reticulum calcium (SERCA) inhibitors, curcumin and thapsigargin, have been reported to effectively correct the CF ion transport defects observed in the ΔF508 CF mice. We investigated the effect of these compounds in human airway epithelial cells to determine if they could induce ΔF508 CFTR maturation, and Cl− secretion. We also used Baby Hamster Kidney cells, heterologously expressing ΔF508 CFTR, to determine if SERCA inhibitors could interfere with the interaction between calnexin and CFTR and thereby correct the ΔF508 CFTR misfolding defect. Finally, at the whole animal level, we tested the ability of curcumin and thapsigargin to (1) induce Cl− secretion and reduce hyperabsorption of Na+ in the nasal epithelia of the ΔF508 mouse in vivo, and (2) induce Cl− secretion in intestine (jejunum and distal colon) and the gallbladder of the ΔF508 CF mouse. We conclude that curcumin and thapsigargin failed to induce maturation of ΔF508 CFTR, or induce Cl− secretion, as measured by biochemical and electrophysiologic techniques in a variety of model systems ranging from cultured cells to in vivo studies.

Previous studies in native tissues have produced conflicting data on the localization and metabolic fate of WT and ΔF508 cystic fibrosis transmembrane regulator (CFTR) in the lung. Combining immunocytochemical and biochemical studies utilizing new high-affinity CFTR mAbs with ion transport assays, we examined both 1) the cell type and region specific expression of CFTR in normal airways and 2) the metabolic fate of ΔF508 CFTR and associated ERM proteins in the cystic fibrosis lung. Studies of lungs from a large number of normal subjects revealed that WT CFTR protein localized to the apical membrane of ciliated cells within the superficial epithelium and gland ducts. In contrast, other cell types in the superficial, gland acinar, and alveolar epithelia expressed little WT CFTR protein. No ΔF508 CFTR mature protein or function could be detected in airway specimens freshly excised from a large number of ΔF508 homozygous subjects, despite an intact ERM complex. In sum, our data demonstrate that WT CFTR is predominantly expressed in ciliated cells, and ΔF508 CFTR pathogenesis in native tissues, like heterologous cells, reflects loss of normal protein processing.

The breakpoint cluster region protein (Bcr) is a large soluble oligomeric multidomain protein best known because of its involvement in chronic myelogenous leukemia (CML). A chromosomal translocation between its gene and that of the c-abl kinase (`Philadelphia chromosome') plays a major causative role in that malignancy. Thus most attention has been paid to the role of the protein in hemopoietic cells. However, Bcr is also expressed in other cell types including epithelia. Bcr is generally considered to be a cytoplasmic protein but in addition to its kinase and GTPase exchange and activating domains it contains potentially membrane-interacting pleckstrin homology and C2 domains as well as a PDZ-binding C terminus mediating an interaction with a PDZ-domain protein at intercellular junctions of epithelial cells. We have examined the ability of Bcr to interact with other epithelial PDZ proteins and found specific binding to both the apical PDZK1 protein and the Golgi-localized Mint3. The former is important in the organization of several apical functions and the latter in vesicular trafficking in the secretory pathway. Hence these findings extend the interactions and likely signaling impact of Bcr in epithelia from the cytosol to at least these two membrane compartments.