Roya Navab

Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer

The tumor microenvironment strongly influences cancer development, progression, and metastasis. The role of carcinoma-associated fibroblasts (CAFs) in these processes and their clinical impact has not been studied systematically in non-small cell lung carcinoma (NSCLC). We established primary cultures of CAFs and matched normal fibroblasts (NFs) from 15 resected NSCLC. We demonstrate that CAFs have greater ability than NFs to enhance the tumorigenicity of lung cancer cell lines. Microarray gene-expression analysis of the 15 matched CAF and NF cell lines identified 46 differentially expressed genes, encoding for proteins that are significantly enriched for extracellular proteins regulated by the TGF-β signaling pathway. We have identified a subset of 11 genes (13 probe sets) that formed a prognostic gene-expression signature, which was validated in multiple independent NSCLC microarray datasets. Functional annotation using protein–protein interaction analyses of these and published cancer stroma-associated gene-expression changes revealed prominent involvement of the focal adhesion and MAPK signaling pathways. Fourteen (30%) of the 46 genes also were differentially expressed in laser-capture–microdissected corresponding primary tumor stroma compared with the matched normal lung. Six of these 14 genes could be induced by TGF-β1 in NF. The results establish the prognostic impact of CAF-associated gene-expression changes in NSCLC patients.

Integrin α11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells

Integrin α11 (ITGA11/α11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal α11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human α11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WTshIGF2) fibroblasts resulted in a decreased growth rate of A549+WTshIGF2, compared with A549+WT tumors. The results indicate that α11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma–stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth.

MicroRNA-21 (miR-21) Regulates Cellular Proliferation, Invasion, Migration, and Apoptosis by Targeting PTEN , RECK and Bcl-2 in Lung Squamous Carcinoma, Gejiu City, China

Background In South China (Gejiu City, Yunnan Province), lung cancer incidence and associated mortality rate is the most prevalent and observed forms of cancer. Lung cancer in this area is called Gejiu squamous cell lung carcinoma (GSQCLC). Research has demonstrated that overexpression of miR-21 occurs in many cancers. However, the unique relationship between miR-21 and its target genes in GSQCLC has never been investigated. The molecular mechanism involved in GSQCLC must be compared to other non-small cell lung cancers in order to establish a relation and identify potential therapeutic targets. Methodology/Principal Findings In the current study, we initially found overexpression of miR-21 occurring in non-small cell lung cancer (NSCLC) cell lines when compared to the immortalized lung epithelial cell line BEAS-2B. We also demonstrated that high expression of miR-21 could increase tumor cell proliferation, invasion, viability, and migration in GSQCLC cell line (YTMLC-90) and NSCLC cell line (NCI-H157). Additionally, our results revealed that miR-21 could suppress YTMLC-90 and NCI-H157 cell apoptosis through arresting cell-cycle at G2/M phase. Furthermore, we demonstrated that PTEN, RECK and Bcl-2 are common target genes of miR-21 in NSCLC. Finally, our studies showed that down-regulation of miR-21 could lead to a significant increase in PTEN and RECK and decrease in Bcl-2 at the mRNA and protein level in YTMLC-90 and NCI-H157 cell lines. However, we have not observed any remarkable difference in the levels of miR-21 and its targets in YTMLC-90 cells when compared with NCI-H157 cells. Conclusions/Significance miR-21 simultaneously regulates multiple programs that enhance cell proliferation, apoptosis and tumor invasiveness by targeting PTEN, RECK and Bcl-2 in GSQCLC. Our results demonstrated that miR-21 may play a vital role in tumorigenesis and progression of lung squamous cell carcinoma and suppression of miR-21 may be a novel approach for the treatment of lung squamous cell carcinoma.

Photodynamic Molecular Beacon Triggered by Fibroblast Activation Protein on Cancer-Associated Fibroblasts for Diagnosis and Treatment of Epithelial Cancers

Fibroblast activation protein (FAP) is a cell-surface serine protease highly expressed on cancer-associated fibroblasts of human epithelial carcinomas but not on normal fibroblasts, normal tissues, and cancer cells. We report herein a novel FAP-triggered photodynamic molecular beacon (FAP-PPB) comprising a fluorescent photosensitizer and a black hole quencher 3 linked by a peptide sequence (TSGPNQEQK) specific to FAP. FAP-PPB was effectively cleaved by both human FAP and murine FAP. By use of the HEK293 transfected cells (HEK-mFAP, FAP(+); HEK-vector, FAP(-)), systematic in vitro and in vivo experiments validated the FAP-specific activation of FAP-PPB in cancer cells and mouse xenografts, respectively. FAP-PPB was cleaved by FAP, allowing fluorescence restoration in FAP-expressing cells while leaving non-expressing FAP cells undetectable. Moreover, FAP-PPB showed FAP-specific photocytotoxicity toward HEK-mFAP cells whereas it was non-cytotoxic toward HEK-Vector cells. This study suggests that the FAP-PPB is a potentially useful tool for epithelial cancer detection and treatment.

From Melanocyte to Metastatic Malignant Melanoma

Malignant melanoma is one of the most aggressive malignancies in human and is responsible for almost 60% of lethal skin tumors. Its incidence has been increasing in white population in the past two decades. There is a complex interaction of environmental (exogenous) and endogenous, including genetic, risk factors in developing malignant melanoma. 8–12% of familial melanomas occur in a familial setting related to mutation of the CDKN2A gene that encodes p16. The aim of this is to briefly review the microanatomy and physiology of the melanocytes, epidemiology, risk factors, clinical presentation, historical classification and histopathology and, more in details, the most recent discoveries in biology and genetics of malignant melanoma. At the end, the final version of 2009 AJCC malignant melanoma staging and classification is presented.

Abelson Interactor Protein-1 Positively Regulates Breast Cancer Cell Proliferation, Migration, and Invasion

Abelson interactor protein-1 (ABI-1) is an adaptor protein involved in actin reorganization and lamellipodia formation. It forms a macromolecular complex containing Hspc300/WASP family verprolin-homologous proteins 2/ABI-1/nucleosome assembly protein 1/PIR121 or Abl/ABI-1/WASP family verprolin-homologous proteins 2 in response to Rho family-dependent stimuli. Due to its role in cell mobility, we hypothesized that ABI-1 has a role in invasion and metastasis. In the present study, we found that weakly invasive breast cancer cell lines (MCF-7, T47D, MDA-MB-468, SKBR3, and CAMA1) express lower levels of ABI-1 compared with highly invasive breast cancer cell lines (MDA-MB-231, MDA-MB-157, BT549, and Hs578T), which exhibit high ABI-1 levels. Using RNA interference, ABI-1 was stably down-regulated in MDA-MB-231, which resulted in decreased cell proliferation and anchorage-dependent colony formation and abrogation of lamellipodia formation on fibronectin. Down-regulation of ABI-1 decreased invasiveness and migration ability and decreased adhesion on collagen IV and actin polymerization in MDA-MB-231 cells. Additionally, compared with control parental cells, ABI-1 small interfering RNA–transfected cells showed decreased levels of phospho-PDK1, phospho-Raf, phospho-AKT, total AKT, and AKT1. These data suggest that ABI-1 plays an important role in the spread of breast cancer and that this role may be mediated via the phosphatidylinositol 3-kinase pathway. (Mol Cancer Res 2007;5(10):1031–9)

Targeting Focal Adhesion Kinase with Dominant-Negative FRNK or Hsp90 Inhibitor 17-DMAG Suppresses Tumor Growth and Metastasis of SiHa Cervical Xenografts

Focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase and key modulator of integrin signaling, is widely expressed in different tissues and cell types. Recent evidence indicates a central function of FAK in neoplasia where the kinase contributes to cell proliferation, resistance to apoptosis and anoikis, invasiveness, and metastasis. FAK, like other signaling kinases, is dependent on the chaperone heat shock protein 90 (Hsp90) for its stability and proper function. Thus, inhibition of Hsp90 might be a way of disrupting FAK signaling and, consequently, tumor progression. FAK is expressed in high-grade squamous intraepithelial lesions and metastatic cervical carcinomas but not in nonneoplastic cervical mucosa. In SiHa, a cervical cancer cell line with characteristics of epithelial-to-mesenchymal transition, the stable expression of dominant-negative FAK-related nonkinase decreases anchorage independence and delays xenograft growth. FAK-related nonkinase as well as the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin both negatively interfere with FAK signaling and focal adhesion turnover. Short-term 17-dimethylaminoethylamino-17-demethoxygeldanamycin treatment prolongs survival in a SiHa lung metastasis model and chronic administration suppresses tumor growth as well as metastatic spread in orthotopic xenografts. Taken together, our data suggest that FAK is of importance for tumor progression in cervical cancer and that disruption of FAK signaling by Hsp90 inhibition might be an avenue to restrain tumor growth as well as metastatic spread.

Molecular biology of normal melanocytes and melanoma cells

Malignant melanoma is one of the most aggressive malignancies in humans and is responsible for 60–80% of deaths from skin cancers. The 5-year survival of patients with metastatic malignant melanoma is about 14%. Its incidence has been increasing in the white population over the past two decades. The mechanisms leading to malignant transformation of melanocytes and melanocytic lesions are poorly understood. In developing malignant melanoma, there is a complex interaction of environmental and endogenous (genetic) factors, including: dysregulation of cell proliferation, programmed cell death (apoptosis) and cell-to-cell interactions. The understanding of genetic alterations in signalling pathways of primary and metastatic malignant melanoma and their interactions may lead to therapeutics modalities, including targeted therapies, particularly in advanced melanomas that have high mortality rates and are often resistant to chemotherapy and radiotherapy. Our knowledge regarding the molecular biology of malignant melanoma has been expanding. Even though several genes involved in melanocyte development may also be associated with melanoma cell development, it is still unclear how a normal melanocyte becomes a melanoma cell. This article reviews the molecular events and recent findings associated with malignant melanoma.

Modeling of lung cancer by an orthotopically growing H460SM variant cell line reveals novel candidate genes for systemic metastasis

Endobronchial implantation of NCI-H460 cells into the nude rat generates a primary lung tumor with mediastinal lymph node spread, but rarely systemic metastases. We isolated tumor cells from mediastinal nodes, orthotopically reimplanted the cells into nude rats and repeated this four times to derive a cell line, designated H460SM, that spontaneously metastasizes to bone, kidney, brain, soft tissue and contralateral lung. H460SM cells demonstrated higher invasive activity in vitro than parental NCI-H460 cells. Spectral karyotyping revealed a new inversion within 17q and loss of an extra normal copy of chromosome 14 present in parental NCI-H460 cells. Expression profiling of orthotopic primary tumors revealed differential expression of 360 genes. Of these, 173 were represented in the probe set of a 19.2K OCI cDNA microarray previously used to profile the gene expression of surgically resected lung cancer specimens. We have computationally validated clinical importance of these genes by using in silico analysis of 18 cases of pulmonary adenocarcinoma, which were split into two patient groups with markedly different clinical outcome. The model identifies additional novel candidate genes for the progression of lung cancer to systemic metastases and poor prognosis.

Expression of Chicken Ovalbumin Upstream Promoter-Transcription Factor II Enhances Invasiveness of Human Lung Carcinoma Cells

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) plays an essential role in angiogenesis and development. It is differentially expressed in tumor cell lines, but its role in carcinogenesis is largely unknown. We demonstrate here that noninvasive human lung cancer cells become invasive when COUP-TFII was expressed. The expression of extracellular matrix degrading proteinases, such as matrix metalloproteinase 2 and urokinase-type plasminogen activator, was up-regulated in these cells. This finding was confirmed by transduction of different human lung cancer cell lines with COUP-TFII protein and also by using antisense expression. We observed disorganization of actin filaments and focal adhesion kinase phosphorylation in COUP-TFII-transfected human lung cancer cells in addition to the increase in extracellular metalloproteinase activity. These results suggest that COUP-TFII may be considered as a new target for anticancer therapies.