Landon J. Inge

Tumor Response to Combination Celecoxib and Erlotinib Therapy in Non-small Cell Lung Cancer Is Associated with a Low Baseline Matrix Metalloproteinase-9 and a Decline in Serum-Soluble E-Cadherin

Introduction: Cyclooxygenase-2 overexpression may mediate resistance to epidermal growth factor receptor tyrosine kinase inhibition through prostaglandin E2-dependent promotion of epithelial to mesenchymal transition (EMT). Suppression of epithelial markers, such as E-cadherin, can lead to resistance to erlotinib. Prostaglandin E2 down-regulates E-cadherin expression by up-regulating transcriptional repressors, including ZEB1 and Snail. Furthermore, E-cadherin can be modulated by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), promoting tumor invasion and metastasis. Markers of EMT and tumor invasion were evaluated in patient serum from a phase I clinical trial investigating the combination of celecoxib and erlotinib in non-small cell lung cancer (NSCLC) patients. Methods: Samples from 22 subjects were evaluated. Soluble E-cadherin (sEC) was evaluated by enzyme linked immunosorbent assay in patient serum at baseline, week 4, and week 8 of treatment. Other markers of EMT and angiogenesis were evaluated by enzyme linked immunosorbent assay, including MMP-9, TIMP-1, and CCL15. Results: Serum sEC, MMP-9, TIMP-1, and CCL15 levels were determined at baseline and week 8. Patients with a partial response to therapy had a significant decrease in sEC, TIMP-1, and CCL15 at week 8. In patients who responded to the combination therapy, baseline MMP-9 was significantly lower compared with nonresponders (p = 0.006). Conclusions: sEC, MMP-9, TIMP-1, and CCL15 levels correlate with response to combination therapy with erlotinib and celecoxib in patients with NSCLC. A randomized phase II trial is planned comparing erlotinib and celecoxib with erlotinib plus placebo in advanced NSCLC. This study will prospectively assess these and other biomarkers in serum and tumor tissue.

N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: implications for immunotherapy

Prostate-specific membrane antigen (PSMA) is an important biomarker expressed in prostate cancer cells with levels proportional to tumor grade. The membrane association and correlation with disease stage portend a promising role for PSMA as an antigenic target for antibody-based therapies. Successful application of such modalities necessitates a detailed knowledge of the subcellular localization and trafficking of target antigen. In this study, we show that PSMA is expressed predominantly in the apical plasma membrane in epithelial cells of the prostate gland and in well-differentiated Madin-Darby canine kidney cells. We show that PSMA is targeted directly to the apical surface and that sorting into appropriate post-Golgi vesicles is dependent upon N-glycosylation of the protein. Integrity of the microtubule cytoskeleton is also essential for delivery and retention of PSMA at the apical plasma membrane domain, as destabilization of microtubules with nocodazole or commonly used chemotherapeutic Vinca alkaloids resulted in the basolateral expression of PSMA and increased the uptake of anti-PSMA antibody from the basolateral domain. These results may have important relevance to PSMA-based immunotherapy and imaging strategies, as prostate cancer cells can maintain a well-differentiated morphology even after metastasis to distal sites. In contrast to antigens on the basolateral surface, apical antigens are separated from the circulation by tight junctions that restrict transport of molecules across the epithelium. Thus, antigens expressed on the apical plasma membrane are not exposed to intravenously administered agents. The ability to reverse the polarity of PSMA from apical to basolateral could have significant implications for the use of PSMA as a therapeutic target.

α-Catenin overrides Src-dependent activation of β-catenin oncogenic signaling

Loss of α-catenin is one of the characteristics of prostate cancer. The catenins (α and β) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of β-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where β-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, β-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of β-catenin invariably are altered in cancer. Although a wealth of information is available about β-catenin deregulation during oncogenesis, much less is known about how or whether α-catenin regulates β-catenin functions. In this study, we show that α-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of β-catenin. In α-catenin-null prostate cancer cells, reexpression of α-catenin increased cell-cell adhesion and decreased β-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of β-catenin is a major mechanism for decreased β-catenin interaction with E-cadherin in α-catenin-null cells. α-Catenin attenuated the effect of Src phosphorylation by increasing β-catenin association with E-cadherin. We also show that α-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that α-catenin is a key regulator of β-catenin transcriptional activity and that the status of α-catenin expression in tumor tissues might have prognostic value for Src targeted therapy. [Mol Cancer Ther 2008;7(6):1386–97]

Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase β1-subunit

Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.

Na,K-ATPase β-subunit cis homo-oligomerization is necessary for epithelial lumen formation in mammalian cells

Summary Na,K-ATPase is a hetero-oligomer of an &agr;- and a &bgr;-subunit. The &agr;-subunit (Na,K-&agr;) possesses the catalytic function, whereas the &bgr;-subunit (Na,K-&bgr;) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-&bgr;1 transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-&bgr;1 and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-&agr;1. We now provide evidence that knockdown of Na,K-&bgr;1 prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-&bgr;1 or heptad repeat motif mutant of Na,K-&bgr;1, but not by the glycine zipper motif mutant that abrogates Na,K-&bgr;1 cis homo-oligomerization. These studies suggest that Na,K-&bgr;1 cis homo-oligomerization rather than hetero-oligomerization with Na,K-&agr;1 is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.

P3-033: Serum biomarkers predict response to combination celecoxib and erlotinib therapy in advanced non-small cell lung cancer

Serum biomarkers predict response to combination celecoxib and erlotinib therapy in advanced non-small cell lung cancer Reckamp, Karen L.1 Gardner, Brian K.2 Elashoff, David2 Figlin, Robert A.1 Krysan, Kostyantyn2 Dohadwala, Mariam2 Inge, Landon2 Rajasekaran, Ayyappan2 Dubinett, Steven M.2 1 City of Hope, Duarte, CA, USA 2 UCLA, Los Angeles, CA, USA Background: Cyclooxygenase-2 (COX-2) overexpression may mediate resistance to EGFR TK inhibition through prostaglandin E2 (PGE2)-dependent promotion of epithelial to mesenchymal transition (EMT). The suppression of epithelial markers such as E-cadherin can to non-small cell lung cancer (NSCLC) resistance to erlotinib. In addition, PGE2 can downregulate E-cadherin expression by upregulating the transcriptional repressors of E-cadherin, ZEB1 and Snail. These findings suggest that COX-2 inhibition may enhance the efficacy of EGFR TKI therapy in NSCLC, and markers of EMT may be important in predicting response to this combination of targeted therapy. Methods: A phase I, dose escalation trial to was performed investigating the combination of celecoxib and erlotinib in patients with advanced NSCLC. Soluble E-cadherin was evaluated by ELISA in patient serum at baseline and weeks 4 and 8 of treatment. Other markers of COX-2 gene expression and EMT were evaluated by ELISA, including matrix metalloproteinase (MMP)-9, MMP-2, tissue inhibitor of MMP (TIMP1), and CCL15. Results: 22 patients had serum samples available for evaluation. Serum E-cadherin, MMP-9, MMP-2, TIMP1 and CCL15 were analyzed according to best response (PR, SD or PD). We found a significant decrease in soluble E-cadherin between baseline and week 8 in pts with PR when compared to those with SD or PD (p = 0.021). In patients who responded to the combination therapy, baseline MMP-9 was significantly lower compared to non-responders (p = 0.006). MMP-2 and TIMP1 showed no significant change based on patient response, and CCL15 decreased in patients with PR. Conclusions: Soluble E-cadherin, MMP-9 and other downstream markers of COX-2 gene expression may be useful for assessing response to combination celecoxib and erlotinib in pts with advanced NSCLC. A randomized Phase II trial is planned comparing erlotinib and celecoxib with erlotinib plus placebo in advanced NSCLC, to evaluate the efficacy of this combination therapy and to assess these and other biomarkers in both serum and tumor tissue. Supported by Phase One Foundation Award, UCLA Lung Cancer SPORE NCI P50 CA 90388 and GLAVAHS CDA.