Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2fl/fl attenuated the rapid tumour onset promoted by homozygous deletion of p53fl/fl. Accelerated carcinoma and sarcoma tumour formation in p53+/− females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

Background:The transforming growth factor-beta (TGF- β) pathway has been implicated in proliferation, migration and invasion of various cancers. Endoglin is a TGF-β accessory receptor that modulates signalling. We identified Endoglin as an epigenetically silenced tumour-suppressor gene in lung cancer by means of a genome-wide screening approach, then sought to characterise its effect on lung cancer progression.Methods:Methylation microarray and RNA sequencing were carried out on lung cancer cell lines. Epigenetic silencing of Endoglin was confirmed by methylation and expression analyses. An expression vector and a 20-gene expression panel were used to evaluate Endoglin function. Pyrosequencing was carried out on two independent cohorts comprising 112 and 202 NSCLC cases, respectively, and the impact of Endoglin methylation on overall survival (OS) was evaluated.Results:Methylation in the promoter region resulted in silencing of Endoglin, which could be reactivated by demethylation. Increased invasion coupled with altered EMT marker expression was observed in cell lines with an epithelial-like, but not those with a mesenchymal-like, profile when Endoglin was absent. Methylation was associated with decreased OS in stage I but not in stages II–III disease.Conclusions:We show that Endoglin is a common target of epigenetic silencing in lung cancer. We reveal a link between Endoglin silencing and EMT progression that might be associated with decreased survival in stage I disease.

HER2 is over-expressed in around 20-25% of breast cancers. Due to increased cell growth and differentiation signaling resulting from HER2 over-expression, this sub-type of the disease is associated with aggressive tumor progression and poor prognosis. HER2 targeted therapy has improved patient prognosis, however, only a subgroup of patients derives the optimal benefit, while other patients have refractory disease or develop resistance. miRNAs are small non coding RNAs involved in the RNAi machinery. They are often aberrantly expressed in tumors and miRNA profiles have been seen to evolve over the course of treatment implicating them in therapeutic resistance. Methods: miRNA array completed on the Human TaqMan miRNA card. Gene expression profiling performed by Illumina Human HT12 v4.0 array. Differential gene expression used LIMMA1 empirical Bayes statistics module. Adjusted p-values calculated by Benjamini and Hochberg procedure. In silico target prediction used miRWalk. Lapatinib resistant cells generated by culturing cells in increasing doses of lapatinib up to 2µM. IC50 obtained from drug cytotoxicity measured by MTT analyzed by nonlinear regression. Results: A panel of miRNAs differentially expressed in paired lapatinib sensitive/resistant BT-474 HER2 positive breast cancer cells were identified by miRNA array. Three miRNAs, located in the 14q32 region, were selected for further investigation of their role in resistance to HER2-targeted therapy: miR-127-3p, miR-409-3p, and miR-495-3p. RT-qPCR also confirmed upregulation of these miRNAs in HCC1954 and SKBR-3 HER2 positive cells with acquired resistance to lapatinib, linking increased miRNA expression with acquired resistance. To understand the mechanism behind the miRNA expression in resistant cells we explored epigenetic changes between the sensitive and resistant lines. Global methylation reversal revealed upregulation of all miRNAs in the sensitive cells with a further increase upon addition of an HDAC inhibitor. De-methylation had less effect in resistant cells, indicating loss of methylation as a key component in miRNA upregulation in the process of lapatinib resistance. Using differential gene expression analysis in paired lapatinib-sensitive/resistant BT-474 cells combined with in silico analysis we identified putative targets of these miRNAs. BASP1, a c-MYC transcriptional inhibitor, was down-regulated in the BT-474/L resistant cells. Knock-down altered sensitivity to lapatinib, with an 8 fold shift in IC50 towards resistance, indicating reduced expression of the protein could be involved in the resistance machinery. Conclusions: miR-127, miR-409, and miR-495, clustered to the 14q32 region are subject to epigenetic control, which is altered in lapatinib resistant cells. Resulting upregulation of the miRNAs appears to post-transcriptionally regulate genes, such as BASP1, involved in an acquired resistance mechanism to lapatinib in HER2 positive breast cancer cell lines. Citation Format: Juliette Chupin, Ai Nagano, Victoria Haley, Catherine Lenihan, Francesca Cavicchioli, Karen O’Leary, Natasha Sahgal, Cristiana Lo Nigro, Alice Shia, Claude Chelala, Peter Schmid. miRNAs in the 14q32 cluster are involved in lapatinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2017-104

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