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Keratin 19 regulates cell shape and cell-cell adhesion of MCF7 cells while maintaining E-cadherin localization at the cell surface Welcome to my poster. This is Sarah Alsharif, a PhD student from the biology department. I am glad to present the work our lab has been doing in the breast cancer field. In fact, after lung cancer, breast cancer is the second cause of death in women worldwide (1). It is estimated that every 18 seconds, approximately one new case of breast cancer is documented (2). No one dies due to cancer itself. The death is because of metastasis which takes place when cancer cells leave a breast in which they are formed and reach other sites such as the brain or lung. Our lab is interested in investigating the mechanism behind metastasis of breast cancer. Metastasis is associated with what is called epithelial to mesenchymal transition (EMT), the process characterized by loss of cell to cell adhesion and expression of epithelial markers such as keratin intermediate filament proteins, as you can see in the first three images of cells. Those filaments are keratins and they are critical for the shape and for maintaining mechanical integrity of epithelial cells via cell to cell complexes called desmosomes. Among different keratins, keratin 19 (K19) is highly expressed in many types of cancer including breast cancer, and is correlated with a worse prognosis (3). Consistently, K19 expression has been reported to be significantly higher in metastatic breast cancer tumor cells compared to primary tumors (4). The role of K19 on mechanical properties of cancer cells for cell migration and possible impact on metastasis in breast cancer patients is still unknown. Therefore, our main focus is to know how K19 might regulate cell morphology, specifically shape and adhesion, and the consequences of the overall behavior of breast cancer cells. Using CRISPR Cas9, we were successful in knocking out K19 in the MCF7 breast cancer cell line as you can see in the first western blot. We have two clones of KO cells indicated by KO1 and KO2. By going through the main findings, in the first two results blue bars show parental (P) cells where K19 is present, but yellow and red bars illustrate the two clones of K19 KO cells. We can see in the first graph that K19 KO cells are more elongated compared to P. The second result shows the adhesion made by P and KO cells. To assess this, we classified the degree of adhesion into three different degrees. High degree is when cells are attached tightly to each other, low degree is when cells are point-attached to each other, and medium degree is in between high and low. As you can see, K19 KO cells are attached to each other at a lower degree of adhesion compared to P. From the first two results, we can say that K19 maintains epithelial cell shape and tight cell to cell adhesion of MCF7 breast cancer cells. But what is the mechanism of action that might lead to this phenotype? In order to find out we did a wide RNA-sequencing analysis that shows mRNA levels of all proteins expressed in P and KO cells. Surprisingly, we found that most adhesive molecules are upregulated in KO cells including E-cadherin, the classical one involved in adherens junction (AJ). You can see here both K19 KO cells have higher expression of E-cadherin compared to P. By going back to the introduction section, you can see the AJ complex in which there is E-cadherin. Cytoplasmic sites of E-cadherin bind to beta catenin which was found previously to bind to K19 in MCF7, but as you see in the blot total level of beta catenin was not affected by knocking out K19. There is another molecule called plakoglobin which is the only protein that binds to both desmosomes and AJ, and is found to be highly affected and decreased by knocking out K19. This data suggests that K19 regulates E-cadherin and it has an effect on stabilizing the E- cadherin complex at AJ. Therefore, we did IF staining and IP to test this hypothesis, and we found that K19 KO cells have less membrane E-cadherin compared to P cell as you can see in the green signal of the images. Also, we did a functional assay called biotin labeling protein surface to pull down surface protein including E-cadherin, and we see a significant decrease of membrane E-cadherin in KO cells as you can see here in the blot. K19 KO cells may try to compensate for the lack of K19 by expressing more E-cadherin, but most of it is accumulated in cytoplasm not reaching the cell membrane where it functions. Therefore, we tagged endosomes protein with transferrin in red and stained E-cadherin in green, and we see that most of E-cadherin in K19 KO cells is co-localized with early and recycling endosomes compared to P cells. From this result, we can say K19 regulates E-cadherin expression, stability and localization in MCF7. When we re-expressed K19 back into KO cells, we see a significant increase of rounded cell shape and a decrease of elongated cell shape, and this confirmed the phenotype we previously saw. Then, we did a wound healing assay to see the effect of K19 deletion on migration potential of MCF7. You can see here, K19 KO cells closed the wound faster and have higher migration potential compared to P. This data suggests that K19 inhibits migration, but as we mentioned before, K19 in patients correlates with worse prognosis. To understand this better we did many assays, one of which is called soft agar assay that assesses the aggressiveness of cells and shows how they can grow in more challenging conditions like semi-solid media, which in this case is agar. We can see P cells could grow better and bigger compared to KO cells. Those clusters might help P cells to survive longer and have higher anchorage independent growth compared to K19 KO cells. We confirmed that by re-expressing K19 back into the KO cell, and we saw less growth. In short, our data suggests that K19 depletion destabilizes E-cadherin at the AJ complex for decreased cell adhesion and increased cell migration, but it reduces anchorage independent growth of MCF7. This growth might be similar to the growth of breast cancer cells growing in clusters upon metastasis in the circulation of breast cancer patients. So, they can survive better and longer, but more experiments should be conducted in order to investigate this hypothesis in vivo. References: (1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745227/ (2) https://www.nature.com/articles/s41572-019-0111-2 (3) https://www.ncbi.nlm.nih.gov/pubmed/25156534 (4) https://clincancerres.aacrjournals.org/content/18/4/993 .

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