c-Myc suppresses the tumorigenicity of lung cancer cells and down-regulates vascular endothelial growth factor expression.

The c-myc oncogene is frequently amplified in cells grown from lung tumors and has been linked to the malignancy of these cancers. In support of this, c-myc transfection enhances the in vitro proliferation and soft agar cloning of human small cell lung cancer (SCLC) cells. In this study, we surprisingly found that c-myc expression suppressed the formation of tumors by SCLC cells in athymic nude mice. c-myc expression down-regulated the protein and transcript for vascular endothelial growth factor (VEGF) in these SCLC cells, as well as VEGF transcript in rat fibroblasts manipulated for c-myc expression and in liver cells of c-myc-transgenic mice. Finally, bivariate and multivariate analyses demonstrated that the probability of tumor formation from lung cancer cell lines was negatively correlated with the relative expression of c-Myc, positively correlated with the relative expression of VEGF, and that the latent time to tumor formation was increased by the expression of c-Myc and decreased by the expression of VEGF. We hypothesize that, for lung cancer cells, c-Myc suppresses the formation of tumors in vivo by down-regulating VEGF, and that the amplification of c-myc seen in cells grown from lung tumors with a poor prognosis is an artifact of selection for growth in vitro.

Association of the decreased expression of alpha3beta1 integrin with the altered cell: environmental interactions and enhanced soft agar cloning ability of c-myc-overexpressing small cell lung cancer cells.

Small cell lung cancer (SCLC) is a highly invasive and metastatic tumor, and the decreased expression of alpha3beta1 integrin may contribute to its virulence. Alpha3beta1 is a critical integrin for pulmonary development and epithelial integrity, and its reduced expression has been linked to the increased malignancy and invasion of other cancers. The amplification of the c-myc oncogene is seen frequently in relapsed SCLC tumors and is associated with a worsened prognosis. In the present study using a model of SCLC tumor progression, overexpression of c-myc in a classic SCLC cell line, NCI H209, enhanced in vitro features of tumorigenesis, altered the relationships between cell and environment, and markedly down-regulated the expression of the alpha3 integrin subunit at both the transcript and protein levels. This inverse relationship between the expression of the alpha3 integrin subunit and c-myc is mimicked by other c-myc-overexpressing SCLC cell lines. Restoring alpha3 expression in the myc-transfected 209 cells reversed the effects of c-myc: alpha3 transfection increased cell:cell adhesion and reduced soft agar cloning without affecting the in vitro doubling time. The diminished soft agar cloning produced by alpha3 transfection was reversed by an antibody that specifically engages alpha3beta1 integrins, P1B5. These results suggest first, that alpha3beta1 integrin mediates homotypic adhesion of SCLC cells, and second, that unengaged alpha3beta1 integrin suppresses the growth of disaggregated SCLC cells. Thus, the down-regulation of the alpha3 integrin subunit may contribute to the enhanced tumorigenicity of c-myc-overexpressing SCLCs by allowing the growth of tumor cells that have reduced contact with ligand-expressing substratum or cells, a condition that occurs during the growth of the primary tumor, tumor invasion, and metastasis.

The growth inhibitory effect of N1,N12-bis(ethyl)spermine in small cell lung cancer cells is maintained in cells expressing the c-myc and Ha-ras oncogenes.

The N',N"-bis(ethyl) polyamine analogues demonstrate great potential as chemotherapeutic agents for lung cancer. This study examines how the expression of two oncogenes frequently associated with a worsened prognosis in lung cancer, c-myc and mutated ras, as well as the phenotypic transition induced by these genes, affects the sensitivity of small cell lung cancer (SCLC) cells to these polyamine analogues. Treatment with N1,N12-bis(ethyl)spermine (BE-Spm), a representative analogue, depresses polyamine levels and is cytostatic for the NCI H209 classic SCLC cell line. Both the overexpression of c-myc and the expression of oncogenic v-Ha-ras in these cells produce phenotypes that retain sensitivity to this growth inhibition. This sensitivity to BESpm is mediated by distinct pathways in these oncogene-expressing cells. c-myc overexpression markedly increases the expression of ornithine decarboxylase, which is then down-regulated by BESpm. In contrast, v-Ha-ras expression highly induces the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. These findings suggest that the bis(ethyl)polyamine compounds may have broad utility for the treatment of both SCLC and non-SCLC, including those expressing oncogenic c-myc and ras.

Protein kinase C-beta 2 inhibits cycling and decreases c-myc-induced apoptosis in small cell lung cancer cells.

The overexpression of c-myc frequently accompanies the relapse of small cell lung cancer (SCLC) cells and contributes to the poor prognosis of this tumor. In this study, we confirm that transfected c-myc results in decreased homotypic cell aggregation and increased proliferative capacity of SCLC cells when nutrient conditions are adequate. We also find that c-myc contributes to apoptosis when cells are nutrient depleted, and flow cytometry suggests that this enhanced apoptosis is associated with a failure to halt cell cycling, consistent with the experience in other cell types. We previously found that protein kinase C-beta (PKC-beta) expression in NCI H209 (209) SCLC cells increases markedly with c-myc transfection (L. F. Barr et al., Cancer Res., 51: 5514-5519, 1991), and we hypothesized that PKC-beta may mediate some of the effects of c-myc in these cells. We test this hypothesis by transfection of rat PKC-beta 1 and bovine PKC-beta 2 isoforms into 209 cells before and after transfection with c-myc. PKC-beta 1 transfection has no effect on these cells. However, PKC-beta 2 expression has distinct phenotypic consequences. In the parental cells, PKC-beta 2 expression results in increased homotypic cell aggregation and a prolonged doubling time. Furthermore, PKC-beta 2 expression increases the fraction of these cells in G0-G1. In the cells which express a transfected c-myc gene, PKC-beta 2 expression improves the survival of cells in low serum by decreasing myc-induced apoptosis. This effect was associated with, and may be mediated by, a selection for cells in the G0-G1 fraction. We postulate that transfection of c-myc into SCLC cells may select for those expressing the PKC-beta 2 gene because this signal transduction event protects against myc-induced apoptosis.

Cell-substratum interactions mediate oncogene-induced phenotype of lung cancer cells.

In vivo and in vitro studies have linked small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells along a differentiation continuum. The transition of a SCLC toward a NSCLC phenotype is modeled in culture by the simultaneous overexpression of myc and ras genes in cultured SCLC cells. A major phenotypic distinction between SCLC and NSCLC in culture is that SCLC cells usually grow in floating aggregates, whereas NSCLC cells and myc- plus ras-expressing SCLC cells grow as adherent spreading monolayers like other epithelial cells. The present studies examine how myc, ras, cell aggregation, and attachment to laminin may interact to modulate transitions between the SCLC and NSCLC phenotypes. We find that myc-expressing SCLC cells, which normally grow as anchorage-independent cells in plastic flasks, will adhere to laminin and exhibit an epithelial morphology. In this setting, the cells express both NSCLC and SCLC markers, thus resembling a tumor type previously termed NSCLC with neuroendocrine features. Anchorage-dependent SCLC cells simultaneously expressing the myc family and an exogenous ras oncogene move further toward the NSCLC phenotype than the above myc-expressing cells. However, forced suspension of such cells restores the expression of neuroendocrine SCLC features. These studies indicate that cell environment, as much as gene expression events, profoundly affects aspects of the SCLC cell phenotype.

c-myc gene-induced alterations in protein kinase C expression: a possible mechanism facilitating myc-ras gene complementation.

The mechanism(s) by which the c-myc nuclear protein and the membrane-associated ras protein interact to mediate phenotypic changes is unknown. We now find that c-mcy gene expression is associated with alterations in the principal signal transduction pathway through which the ras protein is thought to function. We studied the transcript and protein expression of protein kinase C (PKC) isoforms in a culture line of human small cell lung cancer cells (NCI H209) in which expression of inserted c-myc and Ha-ras genes together, but not alone, causes a transition to a large cell phenotype. In control H209 cells, at the transcript and cell membrane protein levels, PKC-alpha is the dominant PKC species. In this cell line, the expression of an exogenous c-myc gene, but not of a viral Ha-ras gene, causes a 5- to 10-fold increase in the PKC-beta isoform transcript and protein. The insertion of ras into the exogenous myc-expressing 209 cells, in addition to causing phenotypic transition, results in the translocation of the PKC-beta protein from the cytosol to the membrane fraction and a decrease in membrane-associated PKC-alpha. Concomitant with these changes, the increased PKC isoform transcript levels induced by myc alone are completely reversed. These observations suggest that a complex set of PKC transcript and protein alterations, most prominently involving an increased PKC-beta protein level in the cell membrane, a decrease in PKC-alpha protein, and a decrease in all PKC isoform transcripts, may represent a fundamental event(s) for c-myc collaboration with Ha-ras to alter cell phenotype.

Transitions between lung cancer phenotypes--implications for tumor progression.

Progression from a treatment-sensitive to a treatment-resistant tumor state is a virtually universal phenomenon in patients with small-cell lung carcinoma (SCLC). In such individuals, this tumor progression may involve transitions from a SCLC to a non-SCLC lung cancer phenotype. We are investigating the cell and molecular biology aspects of these transitions and have derived a cell culture model of one such change, oncogene-induced transition of SCLC to the large-cell undifferentiated lung cancer phenotype. Here we discuss the potential implication of this model for understanding the cell lineage and molecular events regulating normal bronchial epithelial cell differentiation and their relationships to the histogenesis and behavior of lung cancers.

Factors associated with fatal hemoptysis in cancer patients.

We reviewed the clinical outcome of 58 patients with hemoptysis associated with either a hematologic or solid malignancy. Pulmonary hemorrhage causing death (fatal hemoptysis) occurred in 36 percent of these patients. Fatal hemoptysis occurred in six of eight patients with a hematologic malignancy and a fungal pneumonia. Examination of pathologic specimens from five of these patients revealed fungal invasion of blood vessels. An inflammatory response was absent in three, suggesting that granulocytes are not required for fungal-induced tissue destruction. In patients with a bronchogenic tumor, fatal hemoptysis occurred in six of seven patients with a necrotic squamous cell carcinoma. In contrast, hemoptysis was fatal in only two of ten patients with metastatic lung disease. We conclude that hemoptysis in cancer patients with a fungal pneumonia is an ominous sign that may warrant aggressive interventions to prevent a fatal complication.