ERBB2 Mutation Study Carcinogenesis And Drug Susceptibility

Background and Objectives:Beyond the long known fact that ERBB2is a therapeutic target in breast cancer, it is also related with a variety of other cancer types such as gynecological (ovarian/endometrial) or bladder urothelial carcinomas. From a perspective of both gain of function (GOF) and loss of function (LOF), we focus on oncogenic functional studies of several ERBB2mutations generated from large-scale sequencing data. Tumor cell lines that harbor correspondent mutations are tested againt ERBB2inhibitors for drug sensitivity. Take ERBB2as an example, we connect advancing technology to development and application of clinical targeted therapies in the era of cancer genome explosion, with much work remaining to complete the mutational catalog and altered pathways to realize targeted personalized treatment.Materials and Methods:Part One:Data mining in over a thousand cell lines (CCLE), tumor sampls (TCGA, cBioportal, lung cancer patient data from Shanghai Fudan Cancer Hospital) and data processing via computational tools generate a list of potential oncogenic ERBB2somatic mutations. Site-directed mutagenesis is carried out by either Gateway or Quickchange to insert into pDONR223and pBabepuro vectors. pBabepuro vectors are transfected into293T cells to generate viruses for infecting3T3cells. We harvest the successfully infected3T3cells to plant on soft agar for2weeks then count the colonies, which represent the transforming (oncogenic) ability. Proteins for western blotting are extracted fresh from cell lysate for analysis of ERBB2and p-ERBB2levels. Formation of ERBB2homodimer is also investigated with a specified protocol for clarifying the exact oncogenic mechanism.Part Two:Tumor cell lines that harbor correspondent ERBB2mutations are cultured under ideal condition for drug sensitivity studies. The cell lines we use include ovarian cancer cell lines (OVCAR8, EFO27), endometrial cancer cell lines (HEC1A, HEC1B, MFE319, AN3CA), bladder urothelial cancer cell lines (VMCUB-1,5637) and lung cancer cell lines (H1781, HCC827, DV90,A549, H1563). Growth curve is drawn separately to select the algorithmic growing phase in each cell line with different amount of cells to plate in a96-well plate. Preliminary studies of double ERBB2and Mek inhibitors on cell lines are conducted.Results: Part One:ERBB2mutations S653C, G776V, T733I, D769N, R896H and E1244Q show mild to strong oncogenicity. A new oncogenic mechanism is found in S653C (located in subdomain â…£ of the extracellular domain):homodimers form at this site (adjacent to cell membrane) to activate downstream pathways. Meanwhile, all other mutations located in the kinase domain area activate downstream pathways via direct phosphorylation on the ERBB2tail. No homodimer is found in any of those mutations.Part Two:VMCUB-1cell lines (from bladder urothelial carcinomas) shares a similar sensitivity to afatinib (BIBW-2992) as H1781, the positive control. No cell line shows sensitivity towards lapatinib. HKI-272(the old neratinib) is effective in HEC1B and OVCAR8. Neratinib (newly ordered) kills the negative controls for unknown reasons. MFE319is sensitive to neratinib yet it is hard to draw conclusions.Conclusion:Large-scale sequencing data is less daunting when processed with a combination of different computational tools. Site-directed mutagenesis, vector system and soft agar studies are reliable to determine oncogenicity in intro. Drug sensitivity is good a marker to tell whether or not the targeted mutation is a essential for tumor growth (driver mutation). Both studies conducted in parallel paves the way to in vivo oncogenic studies nicely.