Characterization of PERK-positive polyploid-like breast cancer cells and their association with unfolded protein response signatures

Endoplasmic reticulum stress activates the unfolded protein response (UPR), a pathway linked to breast cancer (BC) progression, chemoresistance, and the formation of polyploid giant cancer cells (PGCCs). In a preliminary study, we identified a small subpopulation of breast cancer cells that were strongly positive for the phospho-PERK antibody in immunohistochemistry (IHC), some of which showed atypical mitosis and PGCC-like morphology. Because PGCCs are associated with tumor aggressiveness and poor outcomes, we hypothesize that the strongly PERK-positive BC cells represent PGCCs or their precursors and are enriched in tumors with active unfolded protein response (UPR) signaling. This study aimed to define the frequency of PERK-positive BC cells in BC, assess their association with UPR activation, and identify transcriptomic and proteomic features linked to tumors containing these cells. FFPE tissues from 131 BC patients (96 TNBC, 35 non-TNBC) were analyzed by IHC using antibodies against phospho-PERK, ATF6, and phospho-IRE1. All samples were collected before treatment. PERK-positive BC cells were defined as tumor cells with strong (3+) cytoplasmic and nuclear phospho-PERK staining. The IHC indicates an association between PERK-positive BC cells and tumors with a TNBC phenotype. These cells represent an heterogeneous population with broad variation in cell morphology and size, and mitosis status. Based on IHC results, 21 tumors (13 TNBC, 8 non-TNBC) were selected for nCounter multiomics, using BC360 and MO protein panel for transcriptomic and proteomic analysis, respectively. Eleven tumors contained abundant PERK-positive BC cells, while ten had few or none. Three normal breast tissues were included as controls. When breast cancer tumors with abundant PERK-positive BC cells were compared with those without, 388 genes were differentially expressed (p-value threshold 0.05), of which 296 had p-values < 0.01. Of them, SLPI, S100A7, SERPINB5, KIT, and PHGDH showed the most significant increase in expression (log fold-change >2.0), while 132 genes, including TFF1, PIP, TFF3, CDCA8, PAX5, IL24, and VEGFD, showed the most significant decrease in expression (log fold-change <-2.0). When BC tumors with abundant PERK+ cells were compared with normal breast tissue, 315 genes were differentially expressed (p-value threshold 0.05), of which 184 had p-values < 0.01. Of them, 28, including SPP1, KIFC1, CDK1, TOP2A, RRM2, CENPF, and MYBL2 had a log fold-change >2.0, while 94 genes, including APOD, ZBTB16, PDK4, RELN, TFF3, TIMP4, EGF, and VEGFD had a log fold-change <-2.0. Tumors containing abundant PERK-positive BC cells show clear evidence of unfolded protein response activation and a transcriptomic profile associated with aggressive behavior, metabolic reprogramming, and loss of differentiation. This study was funded by a grant from the Breast Cancer Research Foundation of Alabama (BCRFA) to LPY.