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Loss of cytoplasmic CDK1 predicts poor survival in human lung cancer and confers chemotherapeutic resistance

ABSTRACT: The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early-stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. In these studies, we systematically profiled gene expression in normal (NHBE), immortalized (BEAS-2B) and fully transformed (NNK-BEAS-2B) human bronchial epithelial cells, as well as a non-small cell lung cancer (NSCLC) cell line (H157) from a smoker. Expression profiles that accompany the immortalization and/or transformation of bronchial epithelial cells were generated, and expression of 28 genes was validated by immunoblotting. 4 of them were further evaluated in immunohistochemical analyses of tissue microarrays that contain NSCLC specimens, surrounding non-diseased tissues and non-pulmonary normal tissues. Although all 4 genes were predominantly expressed in tumor tissues, loss of expression of cytoplasmic CDK1 was clinically important because it was associated with a poor prognosis for NSCLC patients. This poor prognostic value may be associated with therapeutic resistance because decreasing levels of cytoplasmic CDK1 in vitro increased resistance to standard chemotherapies used in the treatment of NSCLC, especially microtubule agents where resistance was almost complete. These studies illustrate how a combined microarray approach can facilitate the identification of new, relevant targets in cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE28282 | GEO | 2011/03/31

SECONDARY ACCESSION(S): PRJNA139263

REPOSITORIES: GEO

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Project description:The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early-stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. In these studies, we systematically profiled gene expression in normal (NHBE), immortalized (BEAS-2B) and fully transformed (NNK-BEAS-2B) human bronchial epithelial cells, as well as a non-small cell lung cancer (NSCLC) cell line (H157) from a smoker. Expression profiles that accompany the immortalization and/or transformation of bronchial epithelial cells were generated, and expression of 28 genes was validated by immunoblotting. 4 of them were further evaluated in immunohistochemical analyses of tissue microarrays that contain NSCLC specimens, surrounding non-diseased tissues and non-pulmonary normal tissues. Although all 4 genes were predominantly expressed in tumor tissues, loss of expression of cytoplasmic CDK1 was clinically important because it was associated with a poor prognosis for NSCLC patients. This poor prognostic value may be associated with therapeutic resistance because decreasing levels of cytoplasmic CDK1 in vitro increased resistance to standard chemotherapies used in the treatment of NSCLC, especially microtubule agents where resistance was almost complete. These studies illustrate how a combined microarray approach can facilitate the identification of new, relevant targets in cancer. Fluorescently labeled cDNA were synthesized from 100 microgram RNA by oligo(dT)-primed reverse transcription in the presence of Cy3- or Cy5-dUTP (Amersham Bisciences, Piscataway, NJ). Purified Cy3/Cy5-labelled probes were combined and hybridized in the presence of 2x Denhart's solution, 3.2x saline sodium citrate (SSC), and 0.5% sodium dodecyl sulfate (SDS) in a humidified chamber at 65°C overnight. Prior to scanning (Agilent Technologies, Foster City, CA), slides were successively washed at 22°C in 0.5x SSC/0.1% SDS for 2 min, 0.5x SSC/0.01% SDS for 2 min, and 0.06x SSC for 2 min. Image analyses were performed with the IPLab software (Fairfax, VA). The reference cell (NHBE) was included in every individual hybridization to allow for normalization of each clone’s expression relative to the reference for each cell line (BEAS-2B, NNK-BEAS-2B or H157). A self-to-self hybridization with dye reversal was performed to exclude preferential differences in probe labeling (not included here). Every sample was labeled with Cy5 and Cy3 and hybridized twice. The two fluorescent images (red and green channels) obtained from the scanner constituted the intensity raw data from which differential gene expression ratios and quality control values were calculated. All data were entered into a relational database, using the FileMaker Pro 5 software (Santa Clara, CA). Genes were identified as differentially regulated only if corrected red/green hybridization signals differed by at least two-fold. The genes in each group were alphabetically listed and the ratios of BEAS-2B/NHBE, NNK-BEAS-2B/NHBE or H157/NHBE were graphically visualized by Cluster and TreeView programs (http://rana.lbl.gov/EisenSoftware.htm). These methods fulfilled the MIAME criteria (http://www.mged.org/miame).

Project description:Hyperactivation of the PI3K/AKT pathway is observed in most NSCLCs, promoting proliferation, migration and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of the positive regulator phosphatydil-inositol-3â phosphate kinase (PIK3CA) and/or mutations of AKT1 itself. However, whereas the effects of AKT activation on mRNAs and proteins in the cell are fairly clear, the role of miRNAs as downstream targets of activated PI3K/AKT signalling is still poorly defined so far. To identify miRNAs that are targets of constitutive signalling of PI3K/AKT in lung cancer cells, we performed miRNA profiling of human lung epithelial cells expressing active mutant AKT1 (E17K), active mutant PI3KCA (E545K) or that are silenced for PTEN. We identified 28 differentially expressed miRNAs (8 up-regulated, 20 down-regulated) that were common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells. Among the 8 up-regulated miRNAs that were common to all the alterations, the miRNA most consistently up-regulated by activation of the PI3K/AKT pathway in BEAS-2B cells was miR-196a. The results reported here demonstrate that miR-196a acts as oncogene downstream the PI3K/AKT pathway, mediating the proliferative, pro-migratory and tumorigenic activity of this pathway in NSCLC cells by targeting FoxO1 and p27 expression. By adoptive expression of miR-196a in BEAS-2B cells, we demonstrated that miR-196a stimulates anchorage-dependent and -independent proliferation, migration and tumorigenicity in BEAS-2B cells. On the other hand, by use of antimir-196a in NCI-H460 cells to silence the endogenous expression of miR-196a, we demonstrate that miR-196a plays a pivotal role in mediating the stimulation of proliferation, migration and tumorigenicity induced by aberrant activation of PI3K/AKT signalling. Accordingly, we found that miR-196a was significantly overexpressed in human NSCLC-derived cell lines (n=6) and primary lung cancer samples (n=28). Finally, based on predicted binding sites for miR-196a by microRNA analysis software, we found that miR-196a affects protein levels of FoxO1 and p27 in NSCLC cells.

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Loss of cytoplasmic CDK1 predicts poor survival in human lung cancer and confers chemotherapeutic resistance

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