Breast Ductal Carcinoma In Situ, MCF10DCIS, and the Proprotein Convertase PCSK5

Ductal carcinoma in situ (DCIS) is a premalignancy characterized by the hyperproliferation of breast ductal cells without invasion into the surrounding tissue. The preeminent model for estrogen receptor-negative DCIS is the MCF10DCIS cell line; MCF10DCIS is a member of the MCF10 series of cell lines, which were serially derived from a single patient sample and represent the progression from normal breast to invasive ductal carcinoma. MCF10 is used for in vitro, 3D spheroid, and intraductal xenograft research and underlies impactful studies of DCIS malignancy, metastasis, metabolism, and mechanotransduction. The TGFβ-superfamily ligand GDF11 is important for the maintenance of epithelial properties in MCF10DCIS spheroids and xenografts. GDF11 is synthesized as an inactive protein precursor and is activated by proprotein convertase PCSK5; in triple-negative breast cancer, GDF11 activity is lost due to PCSK5 silencing. The heterozygous PCSK5 mutation M452I arose during derivation of the MCF10 series but is not documented in breast or any other type of cancer.

Through a carefully-designed set of experiments spanning all complexity levels at which MCF10DCIS is used, we show that PCSK5M452I is not hypermorphic but hypomorphic. Using an optimized in-cell GDF11 maturation assay, we found that overexpressed PCSK5M452I had measurable but significantly decreased activity compared to wildtype PCSK5. Co-expression of wildtype PCSK5 and PCSK5M452I yielded an intermediate activity level. In a PCSK5–/– clone of MCF10DCIS reconstituted with different PCSK5 alleles, PCSK5M452I was mildly defective in anterograde transport. However, the multicellular organization of PCSK5M452I addback cells in 3D matrigel cultures was significantly less compact than wildtype and the growth of intraductal MCF10DCIS xenografts was similarly impaired along with the frequency of comedo necrosis and stromal activation. In the same settings, we found that a PCSK5T288P null allele, which had GDF11-processing activity akin to ‒PCSK5 control, remained in the cis- and particularly the trans-Golgi compartments of the secretory pathway, formed acircular spheroids, and had impaired xenograft growth compared to wildtype PCSK5.

This dissertation reinforces an important role for PCSK5 in the promotion of pro-epithelial phenotype in DCIS. It also reassures the DCIS research community that a PCSK5 mutation unique to MCF10 cell lines is not responsible for the salient characteristics of the MCF10DCIS cell line and xenograft model.