An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells.

The side population (SP) in human lung cancer cell lines and tumors is enriched with cancer stem cells. An endogenous inhibitor of angiogenesis known as tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), characterized for its ability to inhibit matrix metalloproteinases (MMPs), has been shown by several laboratories to impede tumor progression through MMP-dependent or -independent mechanisms. We recently reported that forced expression of TIMP-2, as well as the modified form Ala+TIMP-2 (that lacks MMP inhibitory activity) significantly blocks growth of A549 human lung cancer cells in vivo. However, the mechanisms underlying TIMP-2 antitumor effects are not fully characterized. Here, we examine the hypothesis that the TIMP-2 antitumor activity may involve regulation of the SP in human lung cancer cells. Indeed, using Hoechst dye efflux assay and flow cytometry, as well as quantitative reverse transcriptase-PCR analysis, we found that endogenous TIMP-2 mRNA levels showed a significant inverse correlation with SP fraction size in six non-small cell lung cancer cell lines. In A549 cells expressing increased levels of TIMP-2, a significant decrease in SP was observed, and this decrease was associated with lowered gene expression of ABCG2, ABCB1 and AKR1C1. Functional analysis of A549 cells showed that TIMP-2 overexpression increased chemosensitivity to cytotoxic drugs. The SP isolated from TIMP-2-overexpressing A549 cells also demonstrated impaired migratory capacity compared with the SP from empty vector control. More importantly, our data provide strong evidence that these TIMP-2 functions occur independent of MMP inhibition, as A549 cells overexpressing Ala+TIMP-2 exhibited identical behavior to those overexpressing TIMP-2 alone. Our findings provide the first indication that TIMP-2 modulates SP phenotype and function, and suggests that TIMP-2 may act as an endogenous suppressor of the SP in human lung cancer cells.

Bronchiolization of the alveoli in lung cancer: pathology, patterns of differentiation and oncogene expression.

We examined the incidence and association of bronchiolization of the alveoli with non-small cell lung cancer in lung resection specimens from 2 patient groups: those with non-small cell lung cancer and those diagnosed with a variety of non-neoplastic lung conditions. We observed marked variation in bronchiolization of the alveoli morphology ranging from normal to severely atypical and developed a classification scheme based on growth pattern, cell number and cytologic criteria. Patterns of differentiation, proliferation and growth factor receptor and oncogene expression were studied using immuno-histochemical and in situ hybridization techniques. While low-grade (0-I) bronchiolization of the alveoli lesions demonstrated markers similar to normal bronchiolar epithelium, a significant decrease in the Clara cell 10 kDa protein and tubulin and an increase in surfactant protein-A expression were observed in high-grade (II-III) lesions. Focal p53 expression was detected in 2 high-grade lesions, while c-myc mRNA and cJun protein were observed in all grades. No correlation was observed between bronchiolization of the alveoli incidence and histologic tumor type. A comparison of marker expression in lesions and tumors from the same case revealed a negative correlation between cytokeratin-14 and c-erbB-2 immuno-reactivity. Only one bronchialization of the alveoli lesion was found in the non-neoplastic patient group. We conclude that up to 12% of non-small cell lung cancer resection specimens contain bronchiolization of the alveoli lesions which exhibit altered morphology and patterns of differentiation.