Sanyal, Bharati

Lung cancer is the leading cause of cancer‐related death inthe United States. Pulmonary typical carcinoid (TC) cancers are part of a spectrum of neuroendocrine lung cancers which includes small cell lung cancer (SCLC). Besides surgery, there are limited treatment options available for patients with these cancers, emphasizing the need for the development of new treatments. The c‐erbB family of receptor tyrosine kinases (ErbB1‐4) mediate cellular responses to growth factors and interact with downstream signaling pathways to promote tumorigenesis through abnormal cell proliferation. ErbB1 (EGFR) has received considerable attention in NSCLC since somatic mutations in the tyrosine kinase domain correlated with responsiveness to gefitinib. In NSCLC, breast, and ovarian cancer, ErbB2/ErbB3 heterodimers have been found to be expressed and signal to downstream pathways such as ERK. ErbB4 has been shown to promote proliferation or differentiation of neural tumors (ependymoma, gliomas, and meningiomas), ovarian cancer, and osteosarcoma. We recently examined the expression of ErbB receptors on TC cancers by quantitative real‐time RT‐PCR and immunoblotting. TCs have high expression of ErbB3 with moderate expression of ErbB1 and ErbB4 and lack expression of ErbB2. This contrasts with atypical carcinoids and SCLC which have high expression of ErbB4 with minimal expression of ErbB3 and lack expression of ErbB1 and ErbB2. We believe that investigation of ErbB signaling in pulmonary carcinoids will lead to new insights into the biology of these cancers, the understanding of which could lead to the development of novel treatments. Supported by funds from Mayo Foundation.

The cellular differentiation of Pneumocystis carinii (PC) from the trophic forms to the multinucleated cyst is critical to understand the pathogenesis of this fungus. Still the mechanism of life cycle regulation is not well known. In fungi closely related to PC, a mitogen‐activated protein kinase (MAPK) cascade regulates these processes. Conjugation between two haploid cells occurs after pheromones bind to their respective pheromone receptors on cells of opposite mating types. We previously reported a functional analysis of the PCSTE3 pheromone receptor of PC, and now we report characteristics of the cognate receptor, PCSTE2. We cloned the full‐length 1065‐bp cDNA for PCSTE2, of which the translated open‐reading frame encodes a protein of 354 amino acids with greatest homology to the fungal pheromone receptors from S. cerevisiae Ste2 and S. pombe Mam2. Computer prediction modeling of PCSTE2 reveals seven transmembrane domains characteristic of fungal pheromone receptors. Similar to the S. cerevisiae alpha factor receptor Ste2, PCSTE2 has three extracellular domains implicated in ligand binding. An antibody made against the extracellular domain of PCSTE2 identified PCSTE2 receptor expression on a subpopulation of PC organisms in infected rat lung tissue but not normal uninfected tissue. We expressed PCSTE2 in S. cerevisiae Ste2 knockout yeast for functional characterization. Our identification of both PCSTE2 and PCSTE3 pheromone receptors may help to elucidate mechanisms of PC life cycle regulation and potential new therapeutics for treating PC pneumonia through alterations of MAPK signaling. Supported by NIH grant 2R01 AI‐48409 to CFT.

AbstractPurpose: This study aimed to investigate the expression of the ErbB family of receptor tyrosine kinases in pulmonary typical carcinoid and atypical carcinoid tumors and to understand the role of epidermal growth factor receptor (EGFR) signaling in pulmonary carcinoid tumor proliferation.Experimental Design: Surgically resected typical carcinoid (n = 24) and atypical carcinoid (n = 7) tumor tissues were analyzed by immunohistochemical staining for EGFR, ErbB2, ErbB3, and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene and the KRAS gene was carried out. Biochemical analysis of lung carcinoid cell lines was used to investigate EGFR signal transduction and response to erlotinib inhibition.Results: The analysis showed that 45.8% of typical carcinoid and 28.6% of atypical carcinoid tumors express EGFR, 100% of the tumors lack expression of ErbB2, and 100% have moderate to intense staining for ErbB3 and ErbB4. Sequencing of tumor DNA of exons 18 to 21 of the EGFR gene revealed the absence of tyrosine kinase domain mutations in these tumors. Instead, 80.6% tumors harbored a synonymous single nucleotide polymorphism in exon 20. Because EGFR and KRAS mutations tend not to be present at the same time, we sequenced the KRAS gene from pulmonary carcinoid tumor DNA and found that 100% were wild-type. Using a lung carcinoid cell line that expresses EGFR, we found that erlotinib reduced proliferation by inhibiting EGFR signal transduction.Conclusions: Our findings suggest clinical potential for the use of EGFR inhibitors in the treatment of patients with pulmonary carcinoid tumors, particularly for patients with EGFR-positive pulmonary carcinoid tumors not amenable to surgical resection.

Abstract Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional antimycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel antimycobacterial treatment. The cytochrome P450 enzyme, CYP51, catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function, leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC that functions as a lanosterol 14α-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and electron paramagnetic resonance spectroscopy, and determine the minimum inhibitory concentrations (MICs) of econazole, ketoconazole, itraconazole, fluconazole, and voriconazole against MAC. Furthermore, we demonstrate that econazole has a MIC of 4 μg/ml and a minimum bacteriocidal concentration of 4 μg/ml, whereas ketoconazole has a MIC of 8 μg/ml and a minimum bacteriocidal concentration of 16 μg/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.