PCSK5^M452Iis a recessive hypomorph exclusive to MCF10DCIS.com cells

The most widely used cell line for studying ductal carcinoma in situ (DCIS) premalignancy is the transformed breast epithelial cell line,<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://MCF10DCIS.com">MCF10DCIS.com</ext-link>. During its original clonal isolation and selection,<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://MCF10DCIS.com">MCF10DCIS.com</ext-link>acquired a heterozygous M452I mutation in the proprotein convertase PCSK5, which has never been reported in any human cancer. The mutation is noteworthy because PCSK5 matures GDF11, a TGFβ-superfamily ligand that suppresses progression of triple-negative breast cancer. We asked here whether PCSK5^M452Iand its activity toward GDF11 might contribute to the unique properties of<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://MCF10DCIS.com">MCF10DCIS.com</ext-link>. Using an optimized in-cell GDF11 maturation assay, we found that overexpressed PCSK5^M452Iwas measurably active but at a fraction of the wildtype enzyme. In aPCSK5^−/−clone of<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://MCF10DCIS.com">MCF10DCIS.com</ext-link>reconstituted with different PCSK5 alleles, PCSK5^M452Iwas mildly defective in anterograde transport. However, the multicellular organization of PCSK5^M452Iaddback cells in 3D matrigel cultures was significantly less compact than wildtype and indistinguishable from a PCSK5^T288Pnull allele. Growth of intraductal<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://MCF10DCIS.com">MCF10DCIS.com</ext-link>xenografts was similarly impaired along with the frequency of comedo necrosis and stromal activation. In no setting did PCSK5^M452Iexhibit gain-of-function activity, leading us to conclude that it is hypomorphic and thus compensated by the remaining wildtype allele in<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://MCF10DCIS.com">MCF10DCIS.com</ext-link>.