Mathias Gugger

miR-15a and miR-16 Are Implicated in Cell Cycle Regulation in a Rb-Dependent Manner and Are Frequently Deleted or Down-regulated in Non–Small Cell Lung Cancer

MicroRNAs (miRNA) are negative regulators of gene expression at the posttranscriptional level, which are involved in tumorigenesis. Two miRNAs, miR-15a and miR-16, which are located at chromosome 13q14, have been implicated in cell cycle control and apoptosis, but little information is available about their role in solid tumors. To address this question, we established a protocol to quantify miRNAs from laser capture microdissected tissues. Here, we show that miR-15a/miR-16 are frequently deleted or down-regulated in squamous cell carcinomas and adenocarcinomas of the lung. In these tumors, expression of miR-15a/miR-16 inversely correlates with the expression of cyclin D1. In non-small cell lung cancer (NSCLC) cell lines, cyclins D1, D2, and E1 are directly regulated by physiologic concentrations of miR-15a/miR-16. Consistent with these results, overexpression of these miRNAs induces cell cycle arrest in G(1)-G(0). Interestingly, H2009 cells lacking Rb are resistant to miR-15a/miR-16-induced cell cycle arrest, whereas reintroduction of functional Rb resensitizes these cells to miRNA activity. In contrast, down-regulation of Rb in A549 cells by RNA interference confers resistance to these miRNAs. Thus, cell cycle arrest induced by these miRNAs depends on the expression of Rb, confirming that G(1) cyclins are major targets of miR-15a/miR-16 in NSCLC. Our results indicate that miR-15a/miR-16 are implicated in cell cycle control and likely contribute to the tumorigenesis of NSCLC.

Gastrin-releasing peptide receptors in non-neoplastic and neoplastic human breast.

The regulatory peptide gastrin-releasing peptide (GRP) may play a role in human cancer as a stimulatory growth factor. To understand the potential role of GRP in human breast cancer, we have evaluated GRP receptor expression in human non-neoplastic and neoplastic breast tissues and in axillary lymph node metastases, using in vitro receptor autoradiography on tissue sections with [(125)I]Tyr(4)-bombesin and with [(125)I]D-Tyr(6), beta Ala(11), Phe(13), Nle(14)-bombesin(6-14) as radioligands. GRP receptors were detected, often in high density, in neoplastic epithelial mammary cells in 29 of 46 invasive ductal carcinomas, in 11 of 17 ductal carcinomas in situ, in 1 of 4 invasive lobular carcinomas, in 1 of 2 lobular carcinomas in situ, and in 1 mucinous and 1 tubular carcinoma. A heterogeneous GRP receptor distribution was found in the neoplastic tissue samples in 32 of 52 cases with invasive carcinoma and 12 of 19 cases with carcinoma in situ. The lymph node metastases (n = 33) from those primary carcinomas expressing GRP receptors were all positive, whereas surrounding lymphoreticular tissue was negative. GRP receptors were also present in high density but with heterogeneous distribution in ducts and lobules from all available breast tissue samples (n = 23). All of the receptors corresponded to the GRP receptor subtype of bombesin receptors, having high affinity for GRP and bombesin and lower affinity for neuromedin B. All tissues expressing GRP receptors were identified similarly with both radioligands. These data describe not only a high percentage of GRP receptor-positive neoplastic breast tissues but also for the first time a ubiquitous GRP receptor expression in nonneoplastic human breast tissue. Apart from suggesting a role of GRP in breast physiology, these data represent the molecular basis for potential clinical applications of GRP analogs such as GRP receptor scintigraphy, radiotherapy, or chemotherapy.

Expression profiling of primary non-small cell lung cancer for target identification.

Using a panel of cDNA microarrays comprising 47 650 transcript elements, we have carried out a dual-channel analysis of gene expression in 39 resected primary human non-small cell lung tumours versus normal lung tissue. Whilst ∼11 000 elements were scored as differentially expressed at least twofold in at least one sample, 96 transcripts were scored as over-represented fourfold or more in at least seven out of 39 tumours and 30 sequences 16-fold in at least two out of 39 tumours, including 24 transcripts in common. Transcripts (178) were found under-represented fourfold in at least seven out of 39 tumours, 31 of which are under-represented 16-fold in at least two out of 39 lesions. The relative expression levels of representative genes from these lists were analysed by comparative multiplex RT–PCR and found to be broadly consistent with the microarray data. Two dramatically over-represented genes, previously designated as potential tumour suppressors in breast (maspin) and lung and breast (S100A2) cancers, were analysed more extensively and demonstrate the effectiveness of this approach in identifying potential lung cancer diagnostic or therapeutic targets. Whilst it has been reported that S100A2 is downregulated in NSCLC at an early stage, our microarray, cmRT–PCR, Western and immunohistochemistry data indicate that it is strongly expressed in the majority of tumours.

Maspin - the most commonly-expressed gene of the 18q21.3 serpin cluster in lung cancer - is strongly expressed in preneoplastic bronchial lesions.

Maspin, SCCA1/2 and hurpin were identified by cDNA microarray analyses as dramatically differentially expressed transcripts in primary non-small cell lung cancer (NSCLC). These sequences are located within a 10-gene serpin cluster on 18q21.3. Using comparative RT–PCR, we have investigated the expression of each of these serpins, including their flanking loci, in an independent NSCLC series. Whereas six of the genes (maspin, SCCA1, SCCA2, hurpin, megsin and pAI-2) were commonly differentially expressed in primary lesions, each significantly more often in squamous cell tumours, maspin was identified as the most frequently over-represented sequence in both squamous cell carcinoma and adenocarcinoma. Using a well-characterized monoclonal antibody, we have shown strong maspin expression in tumour protein extracts, detected multiple isoforms of the 42 kDa protein and shown that maspin is localized specifically to the tumour cells within neoplastic lesions. In contrast, most cells in non-neoplastic lung tissue appear not to express the gene, with the exception of the multipotent basal epithelial cells that line the bronchial airway. These reserve cells generally show strong predominantly nuclear localization of maspin. Strong nuclear expression of maspin within primary tumour cells is correlated with increased survival (P=0.05) and a longer remission duration (P=0.02) in resectable-staged patients. However, within the airways of cancer patients and somewhat in contrast to this observation, such expression was more frequently detected in the superficial cells of preneoplastic over non-neoplastic epithelia (P<0.0001), consistent with a role for the protein in early neoplasia.

OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention

Here we investigate the expression of OCT4 human lung adenocarcinoma and bronchioloalveolar carcinoma (BAC) tumor biopsies and tumor-derived primary cell cultures. OCT4 has been detected in several human tumors suggesting a potentially critical role in tumorigenesis. We assessed the presence of OCT4 in clinical tumor samples of both adenocarcinoma and BAC at the cellular and transcriptional levels, respectively. Furthermore, we evaluated tumor-derived cell cultures for potential differences in OCT4 expression. Immunohistochemical analysis depicted OCT4 in 2 of 8 adenocarcinoma tumor samples and 3 of 5 BAC tumor samples, with no apparent difference in the degree of expression among the sections examined. These results were validated by transcript analysis. Flow cytometric assessment of 11 adenocarcinoma-derived cell cultures and 3 BAC-derived cell cultures revealed significantly higher OCT4 expression in adenocarcinoma tumors compared to their normal counterparts. This, however, was not observed in the BAC cultures. Comparative studies of OCT4 in adenocarcinoma and BAC tumor cell cultures demonstrated a dramatically higher expression in the former. The expression of OCT4 may represent a specific and effective target for therapeutic intervention in adenocarcinoma and BAC. In addition, the aberrant expression and distribution of OCT4 may indicate important parameters concerning the differences between adenocarcinoma and BAC.

MicroRNA-29b is involved in the Src-ID1 signaling pathway and is dysregulated in human lung adenocarcinoma

The c-Src kinase regulates cancer cell invasion through inhibitor of DNA binding/differentiation 1 (ID1). Src and ID1 are frequently overexpressed in human lung adenocarcinoma. The current study aimed at identifying microRNAs (miRNAs) involved in the Src-ID1 signaling in lung cancer. Incubation of lung cancer cells with the Src inhibitor saracatinib led to the upregulation of several miRNAs including miR-29b, which was the most highly upregulated miRNA with predicted binding to the ID1 3′-untranslated region (UTR). Luciferase reporter assays confirmed direct binding of miR-29b to the ID1 3′-UTR. Expression of miR-29b suppressed ID1 levels and significantly reduced migration and invasion. Expression of antisense-miR-29b (anti-miR-29b), on the other hand, enhanced ID1 mRNA and protein levels, and significantly increased lung cancer cell migration and invasion, a hallmark of the Src-ID1 pathway. The ectopic expression of ID1 in miR-29b-overexpressing cells was able to rescue the migratory potential of these cells. Both, anti-miR-29b and ID1 overexpression diminished the effects of the Src inhibitors saracatinib and dasatinib on migration and invasion. Saracatinib and dasatinib decreased c-Myc transcriptional repression on miR-29b and led to increased ID1 protein levels, whereas forced expression of c-Myc repressed miR-29b and induced ID1. In agreement, we showed direct recruitment of c-Myc to the miR-29b promoter. miR-29b was significantly downregulated in primary lung adenocarcinoma samples compared with matched alveolar lung tissue, and miR-29b expression was a significant prognostic factor for patient outcome. These results suggest that miR-29b is involved in the Src-ID1 signaling pathway, is dysregulated in lung adenocarcinoma and is a potential predictive marker for Src kinase inhibitors.

Distribution of CCK1 and CCK2 Receptors in Normal and Diseased Human Pancreatic Tissue

BACKGROUND & AIMS The localization and functional role of cholecystokinin (CCK) receptor proteins in normal and diseased human pancreas, particularly in ductal pancreatic carcinomas, remain unclear. METHODS Tissue samples of normal human pancreas, chronic pancreatitis, and ductal pancreatic carcinomas were investigated under carefully controlled conditions for expression of CCK1 and CCK2 receptor messenger RNA (mRNA) and proteins using in situ hybridization and in vitro CCK receptor autoradiography by means of subtype-selective analogues. Synaptophysin immunohistochemistry was used concomitantly for optimal identification of islets, nerves, and tumor areas with neuroendocrine features. RESULTS CCK2 receptor mRNA and proteins were found abundantly in human pancreatic islets in normal pancreas and chronic pancreatitis. CCK1 receptor proteins were found occasionally in small-sized pancreatic nerves, whereas acini expressed a low density of CCK2 receptors in a few cases of chronic pancreatitis. Ductal pancreatic carcinomas rarely expressed CCK receptors; a few receptor-positive tumors, often characterized by neuroendocrine differentiation, expressed the CCK2 receptor at the mRNA or protein level. However, the main source of CCK receptors in the pancreatic tumor samples consisted of CCK2-expressing islets and/or CCK1-expressing nerves rather than neoplastic tissue. CONCLUSIONS These data indicate that the presence of CCK receptors in human ductal pancreatic tumor samples is mainly due to CCK2 expression in residual pancreatic islets and CCK1 in pancreatic nerves. Pancreatic acini and ductal pancreatic tumor cells very rarely express CCK2 receptors. These observations suggest that CCK analogues may not be of clinical use to target most of these cancers.

GPR87 Is an Overexpressed G-Protein Coupled Receptor in Squamous Cell Carcinoma of the Lung

Lung cancer is the leading cause of cancer death worldwide. The overall 5-year survival after therapy is about 16% and there is a clear need for better treatment options, such as therapies targeting specific molecular structures. G-protein coupled receptors (GPCRs), as the largest family of cell surface receptors, represent an important group of potential targets for diagnostics and therapy. We therefore used laser capture microdissection and GPCR-focused Affymetrix microarrays to examine the expression of 929 GPCR transcripts in tissue samples of 10 patients with squamous cell carcinoma and 7 with adenocarcinoma in order to identify novel targets in non-small cell lung carcinoma (NSCLC). The relative gene expression levels were calculated in tumour samples compared to samples of the neighbouring alveolar tissue in every patient. Based on this unique study design, we identified 5 significantly overexpressed GPCRs in squamous cell carcinoma, in the following decreasing order of expression: GPR87 > CMKOR1 > FZD10 > LGR4 > P2RY11. All are non-olfactory and GRAFS (glutamate, rhodopsin, adhesion, frizzled/taste2, secretin family) classified. GPR87, LGR4 and CMKOR1 are orphan receptors. GPR87 stands out as a candidate for further target validation due to its marked overexpression and correlation on a mutation-based level to squamous cell carcinoma.

Quantitative expansion of structural genomic alterations in the spectrum of neuroendocrine lung carcinomas.

The pathogenesis and interrelationships of neuroendocrine lung carcinomas are not well understood. Tissue macro‐arrays prepared from surgical resection specimens from 35 patients with typical carcinoid (TC), six with atypical carcinoid (AC), 13 with large cell neuroendocrine carcinoma (LCNEC), and 15 with small cell lung carcinoma (SCLC) were investigated by fluorescence in situ hybridization (FISH) and immunohistochemistry. Hybridizations with locus‐specific DNA probes demonstrated a high incidence of deletion for the tumour suppressor genes p53 and retinoblastoma (Rb), and for the oncogene cyclin D1, comparable in all carcinoma types. Similarly, an increase of DNA copy number for the Her‐2/neu and c‐myc oncogenes was noted in all neoplasms. A more detailed quantitative analysis of the results, however, demonstrated increasing numbers of cells harbouring these genomic alterations, from low‐grade TC to highly malignant SCLC, with the exception of cyclin D1 deletion. Mutations of the p53 and Rb genes, as assayed by immunohistochemical studies, were observed at high incidence in high‐grade carcinomas, compared with a low incidence in the low‐grade carcinomas. Conversely, in all carcinoma types, neither membrane‐bound Her‐2/neu nor nuclear cyclin D1 was detected. It is concluded that structural genomic alterations are frequent in neuroendocrine lung carcinomas and that their occurrence may be underestimated by immunohistochemical studies alone. The quantitative expansion of the Rb, p53, c‐myc, and Her‐2/neu alterations towards high‐grade carcinomas suggests common pathogenetic mechanisms in the spectrum of these neoplasms. Copyright

S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas

S100A2 gene products were shown to be frequently and dramatically over-represented in non-small cell lung cancer (NSCLC) lesions over normal tissue by microarray analysis. We have now analysed an independent series of NSCLC tumours and multiple matched normal bronchial epithelial sites by RT–PCR and immunohistochemistry to investigate: whether this expression pattern can be confirmed and whether elevated expression is associated with tumour histology, clinical outcome or preneoplasia. In this second series, S100A2 was strongly expressed in 76% (35 out of 46) of tumours, more frequently in squamous cell than adenocarcinomas (P<0.002). This strong expression was not related to high-level gene amplification, but was associated in one of five informative cases with an allele-specific imbalance in transcript levels. Most tumours strongly expressed the ΔNp63 transcript, the product of which is a putative regulator of S100A2 transcription and while all but one of the tumours positive for ΔNp63 expressed S100A2, others negative for this regulator also expressed the gene. Contrary to the hypothesis that S100A2 is a tumour suppressor, no somatic mutations were identified in the coding sequence in 44 tumours. Furthermore, an examination of multiple tumour-free epithelial sites from 20 patients showed that strong expression was often associated with increasing levels of disorder in preinvasive bronchial lesions (P<0.0001).