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Even after development of molecular targeting therapies, platinum-based chemotherapy is still a standard care for treatment of locally advanced non-small cell lung cancer (NSCLC). So far, critical molecular markers capable to predict the therapeutic response in NSCLC patients remain undetermined. We here attempted to identify novel biomarker genes for cisplatin (CDDP) for a tailored therapy. Initial screening to explorer association of IC(50) values of CDDP obtained by MTT assay and gene expression levels measured with oligonucleotide microarray and real-time RT-PCR provided 6 candidate genes, namely, NUBPL, C9orf30, ZNF12, TMEM158, GSK3B, and FBLP1 using 9 lung cancer cells consisting of 3 small and 6 NSCLC cells. These 6 genes together with 5 reported biomarkers, i.e., GSTP1, ERCC1, BRCA1, FRAP1, and RRM1, were subjected to a linear regression analysis using 12 NSCLC cell lines including 6 additional NSCLC cells: only FBLP1 and TMEM158 genes showed positive associations with statistical significances (P = .016 and .026, respectively). The biological significance of these genes was explored by in vitro experiments: Knockdown experiments in PC-9/CDDP cells revealed that the reduced expression of TMEM158 significantly decreased the chemo-resistance against CDDP (P

Our previous report demonstrated a good correlation between high telomerase activity of cancer tissues and a poor prognosis of patients with colorectal cancers, except for several cases. To elucidate the additional factors that contribute to patient prognosis, the correlation among the expression levels of telomere binding proteins (TBP), the lengths of telomeres, the lengths of telomere 3'-overhang (3'-OH) and telomerase activity in 106 paired colorectal cancer and corresponding noncancerous mucosa (NCM) specimens were examined. The expression levels of eight TBP genes (TRF1, TRF2, TIN2, TANK1, TANK2, POT1, RAP1 and TPP1) were analyzed. Among the 106 cases, 35 cases had shortened telomeres (7 kb), 15 had shortened 3'-OH (3'-OH length ratio of cancer/NCM0.5) and 88 were classified as telomerase-activated cancers (activity ratio of cancer/NCM2). Comparison between NCM and cancer in each case showed that all TBP except for POT1 were downregulated in cancers. A survival analysis using a Cox proportional hazard model showed that the survival rate of the telomerase-activated cases with shortened 3'-OH and that of telomerase-inactivated cases were significantly better than that of telomerase-activated cases without 3'-OH shortening, that is, restored or maintained 3'-OH (P = 0.018). In the telomerase-activated cancers, the length of 3'-OH was significantly correlated with the expression levels of POT1. Elongation of telomeric overhang by telomerase, which might be regulated by POT1, may contribute to the increase of malignant potential in colorectal cancers.

Prior laboratory prediction of individual drug response is of key importance in esophageal squamous cell carcinoma (ESCC), because of the extremely narrow therapeutic index of chemotherapy. However, very few critical markers have been validated to date for ESCC. We previously demonstrated that simultaneous performance of two different types of comprehensive gene expression analysis might provide a way to identify potent marker genes for drug sensitivity from the expression-sensitivity correlation analysis alone, but the screening method appeared not to be always effective. Therefore, we attempted to identify novel potent marker genes using a new statistical analysis of oligonucleotide microarray expression data, based on a two-dimensional mixed normal model, and selected 3 and 7 novel candidates for 5-fluorouracil (5-FU) and cis-platinum (CDDP), respectively. Interferon induced transmembrane protein 1 (IFITM1) gene alone, being suggested as a key gene of Wnt pathway, was commonly selected in both screening methods. The transfection analyses and siRNA-mediated knock-down experiments revealed that expression of IFITM1 closely related to cellular sensitivity to CDDP. Considering the fact that drug sensitivity is determined by multiple genes, we established the best linear model using quantified expression data of a set of all the selected marker genes including IFITM1, which converted the quantified expression data of ESCC cell lines into an IC50 value of each drug. In the same way, using the representative genes selected in vitro, we developed highly predictive formulae for disease-free survival (DFS) of the CDDP/5-FU combination after curative operation in esophageal cancer patients (R=0.917). A two-dimensional mixed normal model can be a powerful tool to identify novel drug-response determinants, and the IFITM1 gene selected by the statistical method a novel critical biomarker of CDDP response in ESCC.

Esophageal cancer is a highly lethal disease and the optimal therapy remains unclear. Since adjuvant chemotherapy gives a better chance of survival, we attempted to develop a chemosensitivity prediction model to improve individual responses to therapy. Comprehensive gene expression analyses (cDNA and oligonucleotide microarrays) and MTT assay of 8 drugs in 20 KYSE squamous cell carcinoma cell lines were performed to distinguish candidate marker genes whose expression levels reproducibly correlated with cellular drug sensitivities. After confirmation with real-time RT-PCR, we performed multiple regression analyses to develop drug-sensitivity prediction formulae using the quantified expression data of selected marker genes. Using the same sets of genes, we also constructed prediction models for individual clinical responses to 5-FU-based chemotherapy using 18 cases. We selected 5 better marker genes, known as drug sensitivity determinants, identified 9 novel predictive genes for 4 of 8 anticancer drugs [5-FU, CDDP, DOX, and CPT-11 (SN-38)], and developed highly predictive formulae of in vitro sensitivities to the 4 drugs and clinical responses to 5-FU-based adjuvant chemotherapies in terms of overall and disease-free survivals. Our selected genes are likely to be effective drug-sensitivity markers and formulae using the 9 novel genes would provide advantages in prediction.

We attempted to identify potent marker genes using a new statistical analysis and developed a prediction system for individual response to platinum/paclitaxel combination chemotherapy in ovarian cancer patients based on the hypothesis that expression analysis of a set of the key drug sensitivity genes for platinum and paclitaxel could allow us to predict therapeutic response to the combination. From 10 human ovarian cancer cell lines, genes correlative in the expression levels with cytotoxicities of cisplatin (CDDP) and paclitaxel were chosen. We first selected five reliable prediction markers for the two drugs from 22 genes already known as sensitivity determinants and then identified another 8 novel genes through a two-dimensional mixed normal model using oligomicroarray expression data. Using expression data of genes quantified by real-time reverse transcription-PCR, we fixed the best linear model, which converted the quantified expression data into an IC50 of each drug. Multiple regression analysis of the selected genes yielded three prediction formulae for in vitro activity of CDDP and paclitaxel. In the same way, using the same genes selected in vitro, we then attempted to develop prediction formulae for progression-free survival to the platinum/paclitaxel combination. We therefore constructed possible formulae using different sets of 13 selected marker genes (5 known and 8 novel genes): Utility confirmation analyses using another nine test samples seemed to show that the formulae using a set of 8 novel marker genes alone could accurately predict progression-free survival (r = 0.683; P = 0.042). [Mol Cancer Ther 2006;5(3):767–75]

It is widely accepted that activation of telomerase and maintenance of telomeres play central roles in cellular immortalization for most cancer cells. However, they seem to be insufficient for normal human cells. To elucidate critically responsible genes for telomerase mediated cellular immortalization in non-cancerous cells, we explored the genes that are differentially expressed throughout the immortalization process of normal human cells using cDNA microarrays with novel normalization procedures. We found that the number of genes, differentially expressed during cellular immortalization after ectopic expression of telomerase, dramatically increased in a later phase, especially in fibroblasts. We identified 18 and 20 genes/ESTs dysregulated throughout the cellular immortalization processes in fibroblasts and endothelial cells, respectively, but none of them overlapped. Only BGN and COL5A2 were commonly downregulated, except for at early phase in fibroblasts, and a few genes showed controversial expression changes, with regard to previous reports in cancer cells. These findings indicate that normal somatic cells would require cell-type specific events in addition to telomerase activation, and a rare population that eventually experience such events would acquire immortality. The key molecules that distinguish the immortalization mechanisms in cancerous and non-cancerous cells may become crucial targets for anticancer therapy and regenerative therapy.

We developed concise, accurate prediction models of the in vitro activity for 8 anticancer drugs (5-FU, CDDP, MMC, DOX, CPT-11, SN-38, TXL and TXT), along with individual clinical responses to 5-FU using expression data of 12 genes. We first performed cDNA microarray analysis and MTT assay of 19 human cancer cell lines to sort out genes which were correlative in expression levels with cytotoxicities of the 8 drugs; we selected 13 genes with proven functional significance to drug sensitivity from a huge number of potent prediction marker genes. The correlation significance of each was confirmed using expression data quantified by real-time RT-PCR, and finally 12 genes (ABCB1, ABCG2, CYP2C8, CYP3A4, DPYD, GSTP1, MGMT, NQO1, POR, TOP2A, TUBB and TYMS) were selected as more reliable predictors of drug response. Using multiple regression analysis, we fixed 8 prediction formulae which embraced the variable expressions of the 12 genes and arranged them in order, to predict the efficacy of the drugs by referring to the value of Akaike's information criterion for each sample. These formulae appeared to accurately predict the in vitro efficacy of the drugs. For the first clinical application model, we fixed prediction formulae for individual clinical response to 5-FU in the same way using 41 clinical samples obtained from 30 gastric cancer patients and found to be of predictive value in terms of survival, time to treatment failure and tumor growth. None of the 12 selected genes alone could predict such clinical responses.

We previously demonstrated that a new statistical analysis of oligomicroarray expression data based on a two-dimensional mixed normal model could provide a way to identify potent markers for drug sensitivity from the expression-sensitivity correlation analysis alone in esophageal squamous cell carcinoma (ESCC), and the selected genes, 3 for 5-FU and 6 for CDDP, were likely better drug-sensitivity markers, some of which may be the novel drug-response determinants, and developed highly predictive formulae in vitro and in vivo using expression data of a set of the all selected marker genes (100th AACR Annual Meeting). But interestingly, among these selected genes, empty spiracles homolog 2 (EMX2) showed discrepancies between chemo-sensitivity in vitro and patient prognosis in vivo, which led us to focus on the gene. EMX2, the homolog to the ‘empty spiracles’ gene in Drosophila, encodes a homeobox-containing transcription factor, which is critical for central nervous system but has not been reported as a prognostic factor in any cancers so far. So, we attempted to clarify the biological function of EMX2 in cancer cells whether it would be a drug-response determinant or a colony formation capacity determinant through the gene transfection analyses and the soft agar colony formation assay in vitro. Gene transfection analyses demonstrated that the ectopic expression of EMX2 did not correlate with the sensitivity to 5-FU in ESCC cell lines, excluding the former capacity. Meanwhile, we found that in 20 ESCC cell lines, while 9 cell lines with EMX2 little expression showed weak or none colony formation capacity, those with high EMX2 expression showed strong capacity in level dependent manner. Furthermore, colony formation capacity was reduced in EMX2 expression level-dependent manner by siRNA-mediated knockdown in cell lines with high EMX2 expression, suggesting a possible capacity as a colony formation capacity determinant. Using multiple regression analyses, we could develop highly predictive formulae of clinical prognosis in the patients treated with 5-FU/CDDP combination therapy using only 2 selected markers including EMX2, instead of using all the selected genes (Disease free survival: R of the model = 0.798, R of test sample: 0.828; Disease specific survival: R of the model = 0.828, R of test sample: 0.951), and confirmed that expression of EMX2 was a poor prognostic factor in the prediction formulae. These results indicate that EMX2 is the indispensable gene of anchorage-independent growth for ESCC, and high EMX2 expression level is associated with the early recurrence in ESCC. We propose that EMX2 is a novel and potent prognostic maker for ESCC, and elucidation of its molecular mechanism may help to develop new molecular targeting drugs that improve the patient prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1731.