Cetuximab response of lung cancer-derived EGF receptor mutants is associated with asymmetric dimerization.

Kinase domain mutations of the EGF receptor (EGFR) are common oncogenic events in lung adenocarcinoma. Here, we explore the dependency upon asymmetric dimerization of the kinase domain for activation of lung cancer-derived EGFR mutants. We show that whereas wild-type EGFR and the L858R mutant require dimerization for activation and oncogenic transformation, the exon 19 deletion, exon 20 insertion, and L858R/T790M EGFR mutants do not require dimerization. In addition, treatment with the monoclonal antibody, cetuximab, shrinks mouse lung tumors induced by the dimerization-dependent L858R mutant, but exerts only a modest effect on tumors driven by dimerization-independent EGFR mutants. These data imply that different EGFR mutants show differential requirements for dimerization and that disruption of dimerization may be among the antitumor mechanisms of cetuximab.

Suppression of cancer stemness p21-regulating mRNA and microRNA signatures in recurrent ovarian cancer patient samples.

BACKGROUND: Malignant ovarian disease is characterised by high rates of mortality due to high rates of recurrent chemoresistant disease. Anecdotal evidence indicates this may be due to chemoresistant properties of cancer stem cells (CSCs). However, our understanding of the role of CSCs in recurrent ovarian disease remains sparse. In this study we used gene microarrays and meta-analysis of our previously published microRNA (miRNA) data to assess the involvement of cancer stemness signatures in recurrent ovarian disease. METHODS: Microarray analysis was used to characterise early regulation events in an embryonal carcinoma (EC) model of cancer stemness. This was then compared to our previously published microarray data from a study of primary versus recurrent ovarian disease. In parallel, meta-analysis was used to identify cancer stemness miRNA signatures in tumor patient samples. RESULTS: Microarray analysis demonstrated a 90% difference between gene expression events involved in early regulation of differentiation in murine EC (mEC) and embryonic stem (mES) cells. This contrasts the known parallels between mEC and mES cells in the undifferentiated and well-differentiated states. Genelist comparisons identified a cancer stemness signature set of genes in primary versus recurrent data, a subset of which are known p53-p21 regulators. This signature is present in primary and recurrent or in primary alone but essentially never in recurrent tumors specifically. Meta-analysis of miRNA expression showed a much stronger cancer stemness signature within tumor samples. This miRNA signature again related to p53-p21 regulation and was expressed prominently in recurrent tumors. Our data indicate that the regulation of p53-p21 in ovarian cancer involves, at least partially, a cancer stemness component. CONCLUSION: We present a p53-p21 cancer stemness signature model for ovarian cancer. We propose that this may, at least partially, differentially regulate the p53-p21 mechanism in ovarian disease. Targeting CSCs within ovarian cancer represents a potential therapeutic avenue.

ß-Catenin is a useful adjunct immunohistochemical marker for the diagnosis of pulmonary lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare multisystem disease leading to cystic destruction of the lung parenchyma and is associated with abnormal smooth muscle proliferation affecting airways, lymphatics, and blood vessels. LAM occurs sporadically or in association with the tuberous sclerosis complex (TSC). Recent evidence demonstrates the role of aberrant ß-catenin signaling in TSC. To further understand the pathogenesis of LAM and to examine the diagnostic usefulness of ß-catenin, we examined protein expression in 28 pulmonary LAM cases and 10 cases of renal angiomyolipoma resected from patients with sporadic LAM. Immunohistochemical analysis was performed for established markers of LAM cells (HMB45, estrogen receptor [ER]-α, and progesterone receptor [PR]) and ß-catenin. All LAM cases were positive for ß-catenin and demonstrated high specificity with overall immunoreactivity superior to HMB45, ER-α, and PR. Similar expression was demonstrated in renal angiomyolipoma. Our results indicate that ß-catenin is a useful marker of LAM and may be clinically useful in the diagnostic setting.

Analysis of the 10q11 cancer risk locus implicates MSMB and NCOA4 in human prostate tumorigenesis.

Genome-wide association studies (GWAS) have established a variant, rs10993994, on chromosome 10q11 as being associated with prostate cancer risk. Since the variant is located outside of a protein-coding region, the target genes driving tumorigenesis are not readily apparent. Two genes nearest to this variant, MSMB and NCOA4, are strong candidates for mediating the effects of rs109939934. In a cohort of 180 individuals, we demonstrate that the rs10993994 risk allele is associated with decreased expression of two MSMB isoforms in histologically normal and malignant prostate tissue. In addition, the risk allele is associated with increased expression of five NCOA4 isoforms in histologically normal prostate tissue only. No consistent association with either gene is observed in breast or colon tissue. In conjunction with these findings, suppression of MSMB expression or NCOA4 overexpression promotes anchorage-independent growth of prostate epithelial cells, but not growth of breast epithelial cells. These data suggest that germline variation at chromosome 10q11 contributes to prostate cancer risk by influencing expression of at least two genes. More broadly, the findings demonstrate that disease risk alleles may influence multiple genes, and associations between genotype and expression may only be observed in the context of specific tissue and disease states.

Genetic variation in RNASEL associated with prostate cancer risk and progression.

Variation in genes contributing to the host immune response may mediate the relationship between inflammation and prostate carcinogenesis. RNASEL at chromosome 1q25 encodes ribonuclease L, part of the interferon-mediated immune response to viral infection. We therefore investigated the association between variation in RNASEL and prostate cancer risk and progression in a study of 1286 cases and 1264 controls nested within the prospective Physicians' Health Study. Eleven single-nucleotide polymorphisms (SNPs) were selected using the web-based 'Tagger' in the HapMap CEPH panel (Utah residents of Northern and Western European Ancestry). Unconditional logistic regression models assessed the relationship between each SNP and incident advanced stage (T(3)/T(4), T(0)-T(4)/M(1) and lethal disease) and high Gleason grade (>/=7) prostate cancer. Further analyses were stratified by calendar year of diagnosis. Cox proportional hazards models examined the relationship between genotype and prostate cancer-specific survival. We also explored associations between genotype and serum inflammatory biomarkers interleukin-6 (IL-6), C-reactive protein (CRP) and tumor necrosis factor-alpha receptor 2 using linear regression. Individuals homozygous for the variant allele of rs12757998 had an increased risk of prostate cancer [AA versus GG; odds ratio (OR): 1.63, 95% confidence interval (CI): 1.18-2.25), and more specifically, high-grade tumors (OR: 1.90, 95% CI: 1.25-2.89). The same genotype was associated with increased CRP (P = 0.02) and IL-6 (P = 0.05) levels. Missense mutations R462Q and D541E were associated with an increased risk of advanced stage disease only in the pre-prostate-specific antigen era. There were no significant associations with survival. The results of this study support a link between RNASEL and prostate cancer and suggest that the association may be mediated through inflammation. These novel findings warrant replication in future studies.

MYC regulation of a "poor-prognosis" metastatic cancer cell state.

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Regulation of microRNA biosynthesis and expression in 2102Ep embryonal carcinoma stem cells is mirrored in ovarian serous adenocarcinoma patients.

BACKGROUND: Tumours with high proportions of differentiated cells are considered to be of a lower grade to those containing high proportions of undifferentiated cells. This property may be linked to the differentiation properties of stem cell-like populations within malignancies. We aim to identify molecular mechanism associated with the generation of tumours with differing grades from malignant stem cell populations with different differentiation potentials. In this study we assessed microRNA (miRNA) regulation in two populations of malignant Embryonal Carcinoma (EC) stem cell, which differentiate (NTera2) or remain undifferentiated (2102Ep) during tumourigenesis, and compared this to miRNA regulation in ovarian serous carcinoma (OSC) patient samples. METHODS: miRNA expression was assessed in NTera2 and 2102Ep cells in the undifferentiated and differentiated states and compared to that of OSC samples using miRNA qPCR. RESULTS: Our analysis reveals a substantial overlap between miRNA regulation in 2102Ep cells and OSC samples in terms of miRNA biosynthesis and expression of mature miRNAs, particularly those of the miR-17/92 family and clustering to chromosomes 14 and 19. In the undifferentiated state 2102Ep cells expressed mature miRNAs at up to 15,000 fold increased levels despite decreased expression of miRNA biosynthesis genes Drosha and Dicer. 2102Ep cells avoid differentiation, which we show is associated with consistent levels of expression of miRNA biosynthesis genes and mature miRNAs while expression of miRNAs clustering to chromosomes 14 and 19 is deemphasised. OSC patient samples displayed decreased expression of miRNA biosynthesis genes, decreased expression of mature miRNAs and prominent clustering to chromosome 14 but not 19. This indicates that miRNA biosynthesis and levels of miRNA expression, particularly from chromosome 14, are tightly regulated both in progenitor cells and in tumour samples. CONCLUSION: miRNA biosynthesis and expression of mature miRNAs, particularly the miR-17/92 family and those clustering to chromosomes 14 and 19, are highly regulated in both progenitor cells and tumour samples. Strikingly, 2102Ep cells are not simply malfunctioning but respond to differentiation specifically, a mechanism that is highly relevant to OSC samples. Our identification and future manipulation of these miRNAs may facilitate generation of lower grade malignancies from these high-grade cells.

Potentially important microRNA cluster on chromosome 17p13.1 in primary peritoneal carcinoma.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. We characterized the alteration in expression of a select group of microRNAs in primary peritoneal carcinoma relative to matched cases of ovarian serous carcinoma. MicroRNA expression was analysed using semi-quantitative stem-loop RT-PCR on a set of 34 formalin-fixed paraffin-embedded samples. Protein expression of p53 and bcl-2 was quantified in the corresponding tissue microarray. We provide definitive evidence that there is downregulation of a select group of microRNAs in tumours meeting Gynaecological Oncology Group criteria for primary peritoneal carcinoma relative to ovarian serous carcinoma. Specifically, we show decreased p53 expression and downregulation of miR-195 and miR-497 from the microRNA cluster site at chromosome 17p13.1 in primary peritoneal carcinoma relative to ovarian serous carcinoma. miR-195 and miR-497 may have potential roles as tumour-suppressor genes in primary peritoneal tumourigenesis.

Geographical mapping of a multifocal thyroid tumour using genetic alteration analysis & miRNA profiling.

BACKGROUND: Papillary thyroid carcinoma (PTC) frequently presents as multiple tumour-foci within a single thyroid gland or pluriform, with synchronous tumours comprising different histological variants, raising questions regarding its clonality. Among the genetic aberrations described in PTC, the BRAF V600E mutation and ret/PTC activation occur most commonly. Several studies have investigated the genetic alteration status of multifocal thyroid tumours, with discordant results. To address the question of clonality this study examined disparate geographical and morphological areas from a single PTC (classic PTC, insular and anaplastic foci, and tumour cells adjacent to vascular invasion and lymphocytic infiltrate) for the presence of ret/PTC 1 or BRAF mutations. Moreover, we wanted to investigate the consistency of miRNA signatures within disparate areas of a tumour, and geographical data was further correlated with expression profiles of 330 different miRNAs. Putative miRNA gene targets were predicted for differentially regulated miRNAs and immunohistochemistry was performed on tissue sections in an effort to investigate phenotypic variations in microvascular density (MVD), and cytokeratin and p53 protein expression levels. RESULTS: All of the morphological areas proved negative for ret/PTC 1 rearrangement. Two distinct foci with classic morphology harboured the BRAF mutation. All other regions, including the insular and anaplastic areas were negative for the mutation. MiRNA profiles were found to distinguish tumours containing the BRAF mutation from the other tumour types, and to differentiate between the more aggressive insular & anaplastic tumours, and the classic variant. Our data corroborated miRNAs previously discovered in this carcinoma, and additional miRNAs linked to various processes involved in tumour growth and proliferation. CONCLUSION: The initial genetic alteration analysis indicated that pluriform PTC did not necessarily evolve from classic PTC progenitor foci. Analysis of miRNA profiles however provided an interesting variation on the clonality question. While hierarchical clustering analysis of miRNA expression supported the hypothesis that discrete areas did not evolve from clonal expansion of tumour cells, it did not exclude the possibility of independent mutational events suggesting both phenomena might occur simultaneously within a tumour to enhance cancer progression in geographical micro-environments within a tumour.

Altered eIF6 and Dicer expression is associated with clinicopathological features in ovarian serous carcinoma patients.

MicroRNAs are a group of small non-coding RNAs approximately 22 nucleotides in length. Recent work has shown differential expression of mature microRNAs in human cancers. Production and function of microRNAs require coordinated processing by proteins of the microRNA machinery. Dicer and Drosha (RNase III endonucleases) are essential components of the microRNA machinery. Recently, the ribosome anti-association factor eIF6 has also been found to have a role in microRNA-mediated post-transcriptional silencing. We characterized the alterations in the expression of genes encoding proteins of microRNA machinery in ovarian serous carcinoma. Protein expression of eIF6 and Dicer was quantified in a tissue microarray of 66 ovarian serous carcinomas. Dicer, Drosha and eIF6 mRNA expression was analysed using quantitative reverse transcription-PCR on an independent set of 50 formalin-fixed, paraffin-embedded ovarian serous carcinoma samples. Expression profiles of eIF6 and Dicer were correlated with clinicopathological and patient survival data. We provide definitive evidence that eIF6 and Dicer are both upregulated in a significant proportion of ovarian serous carcinomas and are associated with specific clinicopathological features, most notably low eIF6 expression being associated with reduced disease-free survival. The status of eIF6 and proteins of the microRNA machinery may help predict toxicity and susceptibility to future interfering RNA-based therapy.

A molecular expression signature distinguishing follicular lesions in thyroid carcinoma using preamplification RT-PCR in archival samples.

Follicular variant of papillary thyroid carcinoma is a lesion that frequently causes difficulties from a diagnostic perspective in the laboratory. The purpose of this study was to interrogate a cohort of archival thyroid lesions using gene expression analysis of a panel of markers proposed to have utility as adjunctive markers in the diagnosis of thyroid neoplasia and follicular variant of papillary thyroid carcinoma in particular. Laser Capture Microdissection was used to procure pure cell populations for extraction. In addition a novel, multiplex preamplification technique was used to facilitate analysis of multiple targets. The panel comprised: HLA-DMA, HLA-DBQ1, CD74, CSNK1G2, IRF3, KRAS2, LYN, MT1K, MT1X, RAB23, TGFB1 and TOP2A, with CDKN1B as an endogenous control. Expression profiles for each target were generated using TaqMan Real-Time PCR. HLA-DMA, HLA-DQB1, MT1X, CSNK1G2 and RAB23 were found to be differentially expressed (P<0.05) when comparing follicular adenoma and follicular variant of papillary thyroid carcinoma. Comparison of follicular adenoma and follicular thyroid carcinoma groups showed significant differential expression for MT1K, MT1X and RAB23 (P<0.05). Comparison of the papillary thyroid carcinoma group (classic and follicular variants) and the follicular adenoma group showed differential expression for CSNK1G2, HLA-DQB1, MT1X and RAB23 (P<0.05). Finally, KRAS2 was found to be differentially expressed (P<0.05) when comparing the papillary thyroid carcinoma and follicular thyroid carcinoma groups. This panel of molecular targets discriminates between follicular adenoma, papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma and follicular thyroid carcinoma by their expression repertoires. It may have utility for broader use in the setting of fine-needle aspiration cytology and could improve the definitive diagnosis of certain categories of thyroid malignancy.