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Integrative transcriptome-wide analyses reveal critical HER2-regulated mRNAs and lincRNAs in HER2+ breast cancer

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Abstract

Breast cancer is a major health problem affecting millions of women worldwide. Over 200,000 new cases are diagnosed annually in the USA, with approximately 40,000 of these cases resulting in death. HER2-positive (HER2+) breast tumors, representing 20–30 % of early-stage breast cancer diagnoses, are characterized by the amplification of the HER2 gene. However, the critical genes and pathways that become affected by HER2 amplification in humans are yet to be specifically identified. Furthermore, it is yet to be determined if HER2 amplification also affects the expression of long intervening non-coding (linc)RNAs, which are involved in the epigenetic regulation of gene expression. We examined changes in gene expression by next generation RNA sequencing in human tumors pre- and post- HER2 inhibition by trastuzumab in vivo, and changes in gene expression in response to HER2 knock down in cell culture models. We integrated our results with gene expression analysis of HER2+ tumors vs matched normal tissue from The Cancer Genome Atlas. The integrative analyses of these datasets led to the identification of a small set of mRNAs, and the associated biological pathways that become deregulated by HER2 amplification. Furthermore, our analyses identified three lincRNAs that become deregulated in response to HER2 amplification both in vitro and in vivo. Our results should provide the foundation for functional studies of these candidate mRNAs and lincRNAs to further our understanding of how HER2 amplification results in tumorigenesis. Also, the identified lincRNAs could potentially open the door for future RNA-based biomarkers and therapeutics in HER2+ breast cancer.

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Acknowledgments

We would like to thank Maria Sandoval, Jennifer Yori, and Ruth Keri at CWRU Pharmacology for BT474 cells and other reagents; Vinay Varadan at Case Comprehensive Cancer Center and Sheldon Bai at the RNA Center for discussion of bioinformatic analyses; Megan E Forrest, Jessica Sabers, Thomas LaFramboise, and Anthony Wynshaw-Boris for discussion of results.

Conflict of interest

Callie R. Merry, Sarah McMahon, Cheryl L. Thompson, Kristy L.S. Miskimen, Lyndsay Harris, and Ahmad M Khalil declare that there is no conflict of interest to be disclosed.

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Authors and Affiliations

  1. Departments of Genetics and Genome Sciences, Case Western Reserve University, Case Western Reserve University, Cleveland, OH, 44106, USA

    Callie R. Merry, Sarah McMahon & Ahmad M. Khalil

  2. Departments of Biochemistry, Case Western Reserve University, Case Western Reserve University, Cleveland, OH, 44106, USA

    Callie R. Merry & Ahmad M. Khalil

  3. Case Comprehensive Cancer Center, Case Western Reserve University, Case Western Reserve University, Cleveland, OH, 44106, USA

    Cheryl L. Thompson, Kristy L. S. Miskimen, Lyndsay N. Harris & Ahmad M. Khalil

  4. Departments of Medicine, Case Western Reserve University, Case Western Reserve University, Cleveland, OH, 44106, USA

    Lyndsay N. Harris

  5. Departments of Family Medicine and Community Health, Case Western Reserve University, Case Western Reserve University, Cleveland, OH, 44106, USA

    Cheryl L. Thompson

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  1. Callie R. Merry
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  2. Sarah McMahon
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Corresponding author

Correspondence to Ahmad M. Khalil.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10549_2015_3327_MOESM1_ESM.xlsx

Supplementary material 1 (XLSX 10 kb). Supporting file 1. Information regarding clinical trial with trastuzumab in all 13 patients with indication of patients that achieved pathological complete response (pCR) at the end of the trial

10549_2015_3327_MOESM2_ESM.xlsx

Supplementary material 2 (XLSX 11812 kb). Supporting file 2. Differentially expressed mRNAs by RNA-seq in 11 HER2+ breast cancer patients pre- vs post- one dose of trastuzumab. These patients achieved pathological complete response (pCR) at the end of the clinical trial

10549_2015_3327_MOESM3_ESM.xlsx

Supplementary material 3 (XLSX 1961 kb). Supporting file 3. Differentially expressed lincRNAs by RNA-seq in 11 HER2+ breast cancer patients pre- vs post- one dose of trastuzumab. These patients achieved pathological complete response (pCR) at the end of the clinical trial

10549_2015_3327_MOESM4_ESM.xlsx

Supplementary material 4 (XLSX 52 kb). Supporting file 4. Differentially expressed mRNAs by RNA-seq analysis in BT474 cells treated with either negative control (NC) or HER2 siRNAs. Gene expression is calculated as FPKM values

10549_2015_3327_MOESM5_ESM.xlsx

Supplementary material 5 (XLSX 43 kb). Supporting file 5. Differentially expressed lincRNAs by RNA-seq analysis in BT474 cells treated with either negative control (NC) or HER2 siRNAs. Gene expression is calculated as FPKM values

10549_2015_3327_MOESM6_ESM.xlsx

Supplementary material 6 (XLSX 31 kb). Supporting file 6. Pathway analysis of differentially expressed mRNAs (RNA-seq) in BT474 cells treated with either negative control or HER2 siRNAs

10549_2015_3327_MOESM7_ESM.xlsx

Supplementary material 7 (XLSX 7136 kb). Supporting file 7. Differentially expressed mRNAs that are in common between affected mRNAs in response to trastuzumab inhibition of HER2 in clinical trial patients and affected mRNAs due to HER2 depletion in BT474 via siRNAs

10549_2015_3327_MOESM8_ESM.xlsx

Supplementary material 8 (XLSX 1286 kb). Supporting file 8. Differentially expressed lincRNAs that are in common between affected lincRNAs in response to trastuzumab inhibition of HER2 in clinical trial patients and affected lincRNAs due to HER2 depletion in BT474 via siRNAs

10549_2015_3327_MOESM9_ESM.xlsx

Supplementary material 9 (XLSX 4086 kb). Supporting file 9. Information related to HER2+ breast cancer patients from the TCGA cohort that we utilized for gene expression analysis

10549_2015_3327_MOESM10_ESM.xlsx

Supplementary material 10 (XLSX 652 kb). Supporting file 10. Differentially expressed mRNAs (RNA-seq) in HER2+ tumors vs matched normal breast tissue in TCGA cohort. Expression values are calculated as FPKM values

10549_2015_3327_MOESM11_ESM.pdf

Supplementary material 11 (PDF 137 kb). Supporting file 11. Differentially expressed lincRNAs (RNA-seq) in HER2+ tumors vs matched normal breast tissue in TCGA cohort. Expression values are calculated as FPKM values

10549_2015_3327_MOESM12_ESM.xlsx

Supplementary material 12 (XLSX 122 kb). Supporting file 12. Expression values and associated graph of the 35 mRNAs that we identified in all three data sets as responsive to HER2 expression levels

10549_2015_3327_MOESM13_ESM.xlsx

Supplementary material 13 (XLSX 57 kb). Supporting file 13. A complete list of all qPCR primers used in the current study

Supplementary material 14 (XLSX 48 kb). Supporting file 14. Total reads from RNA-seq experiments

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Merry, C.R., McMahon, S., Thompson, C.L. et al. Integrative transcriptome-wide analyses reveal critical HER2-regulated mRNAs and lincRNAs in HER2+ breast cancer. Breast Cancer Res Treat 150, 321–334 (2015). https://doi.org/10.1007/s10549-015-3327-1

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