Breast cancer cells with stem characteristics (abbreviated BCSCs) cause tumour relapse and metastasis. Therapeutic targeting of this cell compartment is challenging as current treatments to eliminate cancer stem-like cells simultaneously affect the normal stem cells of the body. In this project, we proposed to define molecules specifically associated with BCSC, contributing to a better understanding of this crucial cell population.
WP 1: Characterization and targeting of IL6-JAK2-STAT3-S100A8/9 axis in BCSC
Previous studies have indirectly linked HER2 overexpression to the BCSC phenotype but a systematic analysis of this correlation was needed. Our previous results showed that HER2+ tumours have an abnormal activation of the IL6-JAK2-STAT3-S100A8/9 axis that becomes essential for their survival. Therefore, we have analysed the relation between BCSC and HER2 overexpression, as well as with the activation of the identified axis.
It has been described that cell lines contain different amount of stem cells. We selected a panel of breast cancer cell lines with different molecular characteristics and levels of HER2 expression. First we analysed the percentage of BCSC in each cell lines, defined as the ones with a high ALDH activity. Our results show a clear relation between the overexpression of HER2 and the presence of an increased population of stem cells.
We have also confirmed that the HER2+ cells show the activation of the IL6-JAK2-STAT3-S100A8/9. We have worked a syngeneic cell model based in the MCF-10A, in which we have overexpressed the HER2 receptor. By using this model, we have set up the conditions to detect the different components of the pathway in western blot Fluorescent-activated cell sorting (FACS) and using the ImagestreamX (Figure 2). All these techniques confirmed that the overexpression of HER2 triggers the activation of the IL6-JAK2-STAT3-S100A8/9 pathway.
Then, we sorted the HER2+ cell lines according to their ALDH activity and analysed the expression of S100A8 and S100A9 by qRT-PCR. The results showed that both proteins have higher expression in the ALDH+ population
Next, we analysed whether the activation of the IL6-JAK2- STAT3-S100A8/9 activation was essential for BCSC. We used two different approaches: First, we treat BT474 (a cell line that overexpresses HER2) with a JAK2 inhibitor (Ruxolitinib) or a S100A9 inhibitor (Tasquinimod). The inhibition of the pathway resulted in a decreased percentage of stem cells measured as ALDH+ population. Second, we used shRNA to inhibit S100A8 and S100A9 in MCF-10A/HER2 cells and analysed the ability of generating tumorspheres. Again, we observed that the inhibition of the pathway resulted in a decrease in the stem properties of the culture.
During the reported period, we have also set up the conditions for the isolation of CTCs and the detection using the above mentioned antibodies.
WP2: Description of the role of the altered glycosylation patterns in BCSC survival and its contribution to stem phenotype
Aberrant glycosylation occurs in all type of human cancers and it positively correlated with the progression of the tumour being enriched in stem cell-associated proteins. Our aim is to define which of the protein glycosylation components are essential for the BCSC. To reach this goal we have generated the first CRISPR library to target every gene included in the protein glycosylation GO term (GO:0006486). One of the main issues when you do a pooled CRISPR screening is the fact that the LentiCRIPSR V2 vector does not contain the sequence of a fluorescent protein. To bypass this issue we have generated a modified version of the vector including the sequence of tGFP that will facilitate the screening set up. Then, we cloned in this vector the gRNA against the protein glycosylation genes, and validate the equal representation of all of them. This library will be used to perform a pooled screening in a panel of breast cancer cell lines, allowing the identification of the genes that are essential for BCSC and opening the opportunity to new therapeutic approaches
