Keratin 19 regulates cell cycle pathway and sensitivity of breast cancer cells to CDK inhibitors

Abstract

Keratin 19 (K19) belongs to the keratin family of proteins, which maintains structural integrity of epithelia. In cancer, K19 is highly expressed in several types where it serves as a diagnostic marker. Despite the positive correlation between higher expression of K19 in tumor and worse patient survival, the role of K19 in breast cancer remains unclear. Therefore, we ablated K19 expression in MCF7 breast cancer cells and found that K19 was required for cell proliferation. Transcriptome analyses of KRT19 knockout cells identified defects in cell cycle progression and levels of target genes of E2F1, a key transcriptional factor for the transition into S phase. Furthermore, proper levels of cyclin dependent kinases (CDKs) and cyclins, including D-type cyclins critical for E2F1 activation, were dependent on K19 expression, and K19-cyclin D co-expression was observed in human breast cancer tissues. Importantly, K19 interacts with cyclin D3, and a loss of K19 resulted in decreased protein stability of cyclin D3 and sensitivity of cells towards CDK inhibitor-induced cell death. Overall, these findings reveal a novel function of K19 in the regulation of cell cycle program and suggest that K19 may be used to predict the efficacy of CDK inhibitors for treatments of breast cancer.

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

“…In addition, knockdown of E-cadherin abrogated mammosphere formation of MCF7 cells 67 . Therefore, the parallel between K19 and E-cadherin in inhibition of cell migration and promotion of cell proliferation and mammosphere formation 32,66 , together with regulation of E-cadherin localization by K19 suggest that K19 may functionally interact with E-cadherin to promote breast cancer metastasis.…”

“…Cell lines used MCF7 KRT19 KO cells generated using the CRISPR/Cas9 system and MCF7 KRT19 KO cells stably expressing pLenti CMV/TO hygro empty vector or KRT19 have been described previously 32 . For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5'-TCAGTCGACTGGATCCATGGTGAGCAAGGGCGAGG-3' (BamHI site underlined) and 5'-GAAAGCTGGGTCTAGTCAGAGGACCTTGGAGGCAG-3' (XbaI site underlined), following the manufacturer's protocol.…”

“…In our previous work, we had performed RNA-sequencing to identify genes differentially expressed between parental and KRT19 KO cells 32 . Consistent with decreased cell-cell adhesion, KRT19 KO cells expressed reduced levels of plakoglobin, a desmosomal and adherens junction component ( Figure 3A-B) 32 .…”

“…In our previous work, we had performed RNA-sequencing to identify genes differentially expressed between parental and KRT19 KO cells 32 . Consistent with decreased cell-cell adhesion, KRT19 KO cells expressed reduced levels of plakoglobin, a desmosomal and adherens junction component ( Figure 3A-B) 32 . Surprisingly however, a functional enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID) showed that many genes upregulated in KRT19 KO cells were associated with functions in cell membranes, cell adhesions, and cell junctions ( Figure S2).…”

“…MCF7 cells maintain polarized epithelial phenotype with intact cell-cell adhesions made of desmosomes and adherens junctions [29][30][31] . Using MCF7 cells with complete ablation of K19 32 and KRT19 knockout (KO) cells with re-expression of K19, we observed that K19 is required for the epithelial-like cell shape and proper cell-cell adhesion. These events were accompanied by lower levels of plakoglobin but accumulation of E-cadherin in endocytic compartments in the absence of K19.…”