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RNA-seq profiling of trastuzumab or lapatinib sensitive and resistant SKBR3 breast cancer cells

ABSTRACT: RNA-seq profiling of trastuzumab or lapatinib sensitive and resistant SKBR3 breast cancer cells

PROVIDER: PRJNA471825 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7176870_1.fastq.gz	Fastqsanger.gz
	SRR7176870_2.fastq.gz	Fastqsanger.gz
	SRR7176871_1.fastq.gz	Fastqsanger.gz
	SRR7176871_2.fastq.gz	Fastqsanger.gz
	SRR7176872_1.fastq.gz	Fastqsanger.gz

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Project description:HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly. In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells. In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib. Keywords: HER2, breast cancer, resistance, trastuzumab, lapatinib, sorafenib Total RNA was isolated using TRIZOL reagent (Life Technologies, Gathersburg, MD) and quantified by Bioanalyzer 2100 (Agilent Technologies); following the isolation procedure, mRNA was amplified starting from 1μg using MessageAmp II aRNA Amplification kit (Ambion Inc. Austin TX, USA). Ammino-allyl modified nucleotides were incorporated in in vitro transcription step according to the manufacturer’s protocol. Labeling was performed using NHS (N-hydroxysuccinimidyl) ester Cy3 or Cy5 dies (GE Healthcare Europe GMBH, Upsala - Sweden) able to react with the modified RNA. At least 5 μg of mRNA, for each sample, were labeled and purified with columns respectively. The Dye-Swap replication procedure was applied, in order to increase the accuracy of results. Samples were hybridized on 44K glass arrays (Agilent Technologies). Arrays were scanned and the images obtained were analyzed by the Feature Extraction software Agilent (version 9.5) and the text files were then processed using the Bioconductor package Limma (Linear models for microarray analysis).

Project description:Background: Resistance to trastuzumab remains a common challenge to HER-2 positive breast cancer. Up until now, the underlying mechanism of trastuzumab resistance is still unclear. tRNA-derived small non-coding RNAs (tDRs), a new class of small non-coding RNA (sncRNAs), have been observed to play an important role in cancer progression. However, the relationship between tDRs and trastuzumab resistance is still unknown. Methods: We detected the levels of tDRs expression in normal breast epithelial cell lines, trastuzumab-sensitive and -resistant breast cancer cell lines using high-throughput sequencing. qRT-PCR was conducted to validate the differentially expressed tDRs in serums from trastuzumab-sensitive and -resistant patients. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the power of specific tDRs. Progression-free survival (PFS) was analyzed using Cox-regression. Furthermore, Gene Ontology (GO) and pathway analyses indicated the potential mechanism underlying tDR-mediated trastuzumab resistance. Results: Our sequence results showed that tDRs were differentially expressed in the HBL-100, SKBR3, and JIMT-1 cell lines. tDR-1960 and tDR-1969 were found significantly upregulated in trastuzumab-resistant patients compared to sensitive individuals, and the ROC analysis showed that tDR-1960 and tDR-1969 were correlated with trastuzumab resistance. In a multivariate analysis, higher levels of tDR-1960 and tDR-1969 expression were associated with signiﬁcantly shorter PFS in patients with metastatic HER-2 positive breast cancer. Additionally, the GO analysis indicated that tDR-1960 and tDR-1969 were mainly involved in the cellular response to drug, which may partially explain the molecular mechanism underlying trastuzumab resistance in HER-2 positive breast cancer. Conclusion: we comprehensively analyzed tDRs in trastuzumab-sensitive and -resistant breast cancer. Our results suggest that tDR-1960 and tDR-1969 play important roles in trastuzumab resistance. Patients with high levels of tDR-1960 and tDR-1969 expression benefitted less from trastuzumab-based therapy than those that express lower-levels of these tDRs. tDR-1960 and tDR-1969 may be potential biomarkers and intervention targets in the clinical treatment of trastuzumab-resistant breast cancer.

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RNA-seq profiling of trastuzumab or lapatinib sensitive and resistant SKBR3 breast cancer cells

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