Circular HER2 RNA positive triple negative breast cancer is sensitive to Pertuzumab

Abstract

Background Triple negative breast cancer (TNBC) remains the most challenging breast cancer subtype so far. Specific therapeutic approaches have rarely achieved clinical improvements in treatment of TNBC patients and effective molecular biomarkers are largely unknown. Methods We used paired TNBC samples and high throughput RNA sequencing to identify differentially expressed circRNAs. Sucrose gradient polysome fractionation assay, antibody and Mass spectra were used to validate active circRNA translation. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by immunoprecipitation analyses and kinase activity assay. Results Circular HER2 RNA (circ-HER2) encoded a novel protein, HER2–103. Unexpectedly, while HER2 mRNA and protein were barely detected, circ-HER2/HER2–103 was expressed in ~ 30% TNBC clinical samples. Circ-HER2/HER2–103 positive TNBC patients harbored worse overall prognosis than circ-HER2/HER2–103 negative patients. Knockdown circ-HER2 inhibited TNBC cells proliferation, invasion and tumorigenesis in vitro and in vivo, suggesting the critical role of circ-HER2/HER2–103 in TNBC tumorigenicity. Mechanistically, HER2–103 promoted homo/hetero dimerization of epidermal growth factor receptor (EGFR)/HER3, sustained AKT phosphorylation and downstream malignant phenotypes. Furthermore, HER2–103 shared most of the same amino acid sequences as HER2 CR1 domain which could be antagonized by Pertuzumab, a clinical used HER2 antibody. Pertuzumab markedly attenuated in vivo tumorigenicity of circ-HER2/HER2–103 expressing TNBC cells but showed no effects in circ-HER2/HER2–103 negative TNBC cells. Conclusion Our results not only demonstrated that certain TNBCs were not truly ‘HER2 negative’ but also highlighted the clinical implications of Pertuzumab in circ-HER2/HER2–103 expressing TNBC patients.

Regulatory mechanisms, functions, and clinical significance of CircRNAs in triple-negative breast cancer

“…More recently, Li et al confirmed that a newly identified HER2 transcriptional variant, circHER2 , had an open reading frame driven by an IRES and could generate a 103 amino acid protein HER2–103. HER2–103 could promote homo/hetero dimerization of epidermal growth factor receptor (EGFR)/HER3 and sustain AKT phosphorylation and downstream malignant phenotype [ 75 ]. Whith the increasing evidence prove that circRNAs could translate proteins directly [ 72 , 73 , 76 , 77 ], the notion of circRNAs are non-coding RNAs is becoming doubtful.…”

“…Specifically, circRPPH1 , circSEPT9 , circGNB1 , circPGAP3 , circUBE2D2 , circRAD18 , circAGFG1 , circKIF4A , circPLK1 , circUBAP2 , circEPSTI1 , and circGFRA1 were upregulated in both TNBC cells and tissues, and high expression of these circRNAs was able to promote tumor cell proliferation both in vitro and in vivo, and was associated with larger tumor sizes and shorter survival times for TNBC patients [ 29 , 46 , 49 , 51 , 53 , 54 , 57 , 59 , 60 , 63 , 64 , 78 ]. Similarly, circZEB1 , circEIF3M , circHER2 , hsa_circ_0131242 , hsa_circ_0005320 , hsa_circ_069718 , hsa_circ_0058514 , and circTFCP2L1 were overexpressed in TNBC cells and tissues, and they appeared to promote cell proliferation and tumor growth of TNBC [ 55 , 58 , 62 , 75 , 79 – 82 ]. On the contrary, a few circRNAs were identified to have tumor-suppressive effects in TNBC.…”

“…High expression of circSEPT9 , circGNB1 , circAGFG1 , circPGAP3 , circKIF4A , circPLK1 , circANKS1B , circUBAP2 , and ciRS-7 significantly contributed to the invasion and metastasis of TNBC cells both in vitro and in vivo, and were correlated with advanced TNM stage and poor prognosis of TNBC patients [ 45 , 47 , 49 , 51 , 53 , 54 , 57 , 59 , 64 ]. Likewise, circRPPH1 , hsa_circ_0131242 , circEIF3M , circHER2 , circUBE2D2 , circRAD18 , hsa_circ_069718 , hsa_circ_0058514 , and circTFCP2L1 also significantly promoted the migration and invasion capability of TNBC cells in vitro [ 55 , 60 , 62 , 63 , 75 , 78 , 79 , 81 , 82 ]. Conversely, the expression of circFBXW7, circAHNAK1, circTADA2A-E6, and circITCH appeared to be downregulated in TNBCs and was associated with advanced TNM stage and poor survival for TNBC patients [ 48 , 52 , 56 , 74 ].…”

“…Besides, circMTO1 , which is usually downregulated in monastrol-resistant MDA-MB-231 cells, can promote monastrol-induced cytotoxicity by targeting Eg5 and sequestering TRAF4 from binding to the Eg5 gene [ 67 ]. Interestingly, circHER2 , which encodes a novel protein HER2–103, was proved to be expressed in some TNBC samples, and HER2–103-positive TNBC cells were sensitive to Pertuzumab due to HER2–103 shared the same amino acid sequences as the HER2 CR1 domian [ 75 ].…”

“…6 ). High expression of 15 circRNAs ( circSEPT9 , circGNB1 , hsa_circ_0131242 , circHER2 , circPGAP3 , circUBE2D2 , circRAD18 , circAGFG1 , hsa_circ_069718 , circKIF4A , circPLK1 , circANKS1B , circUBAP2 , circEPSTI1 , and circGFRA1 ) was related to worse OS [ 46 , 47 , 49 , 51 , 53 , 54 , 57 , 59 , 63 , 64 , 75 , 78 , 79 , 81 ], indicating that they have carcinogenic effects in TNBC. Increased expression of the 5 circRNAs ( circUSP42 , circFBXW7 , circTADA2A-E6 , circAHNAK1 , and circITCH ) was associated with better OS for TNBC patients [ 48 , 52 , 56 , 74 , 98 ], suggesting that they serve as tumor suppressors.…”