Breast tumour cell subpopulations with expression of the MYC and OCT4 proteins

摘要

TheMYCandOCT4genes are known factors associated with maintaining pluripotency and are linked with a more aggressive course, progression, and resistance to therapy in cancer. Determining the subpopulations of tumour cells expressing the Myc and Oct4 proteins will provide an opportunity to understand which tumour cell subpopulations expressing MYC and OCT4 are associated with metastasis and resistance and which subpopulations can be targeted by anti-MYC and anti-OCT4 therapy. The study included paraffin-embedded tissue from tumours from 27 patients with luminal B breast cancer obtained after neoadjuvant chemotherapy (NACT). Immunofluorescence staining was used to identify subpopulations of tumour cells expressing Myc, Oct4 and Snai2 (Opal (TM) 7-Color Kit (PerkinElmer, Hopkinton, MA). The following tumour cell subpopulations were identified with the Myc and Oct4 proteins and the Snai2 EMT marker: stem/progenitor tumour cells with/without Myc, Oct4 or Snai2 expression; differentiated tumour cells with/without Myc, Oct4 or Snai2 expression; and other nontumour cells (CK7(-)EpCAM(-)CD44(+/-)Myc(+/-)(Oct4, Snai2)(+/-)) within the inflammatory infiltrate in the tumour parenchyma and stroma. The circulating tumour cell subpopulations with Oct4 protein expression in the bloodstream were studied by flow cytometry. It was found that in patients with partial regression (PR) in response to NACT, the frequency of tumour stem cells was 3.6-fold increased (p = 0.038) in the non-EMT state (CK7(+)EpCam(+)CD44(+)Snai2(-)). In patients with metastases, there was a statistically significant 2.5-fold increase in the frequency of differentiated tumour cells with Myc expression (CK7(+)EpCam(+)CD44(-)Myc(+)) and a 2.7-fold increase in the frequency of cells with Oct4 expression (CK7(+)EpCam(+)CD44(-)OCT4(+)). In the next stage, the frequencies of subpopulations with expression of the Oct4 protein and signs of EMT among circulating tumour cells (CTCs) were determined. In patients with metastases, the frequency of tumour stem cells in the EMT state (CD326(+)CD44(+)CD24(-)CD325(+)) (p = 0.015) was more than fourfold increased, and the frequency of progenitor tumour cells with expression of the Oct4 stem protein (CD326(+)CD44(+)CD24(+)Oct4(+)) (p = 0.016) was almost sixfold higher than that in patients without metastases. Nonstem (differentiated) tumour cells with expression of the stemness proteins Myc and Oct4 were present in the breast tumour. Their content was significantly higher in residual tumours after NACT in patients who subsequently developed metastases compared with that in patients without metastases. Such cells are a new in situ marker of metastasis.