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BCSC-ST Report Summary

Project ID: 745808
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - BCSC-ST (Breast cancer stem-like cells specific vulnerabilities: focus in HER2 over-expression and protein glycosylation)

Reporting period: 2017-03-01 to 2019-02-28

Summary of the context and overall objectives of the project

New therapies for breast cancer are based on the molecular differences between cancerous and normal cells and have an increased efficiency and less secondary effects. Unfortunately, a significant number of tumours do not respond to the current treatments. A small population of cells that display stem cell characteristics has been described (Breast Cancer Stem-like Cells; BCSCs) and linked to tumour progression, resistance to treatment and metastasis. Novel therapeutic approaches propose the use of stem cell targeting agents in combination with traditional chemotherapy. However, selective targeting of BCSCs is still challenging, as they are very similar to normal stem cells.
We have worked in two of their characteristics:
1. HER2 Overexpression
Our previous results showed that the axis IL6-STAT3-S100A8/9 is activated and essentaial in breast tumours that overexpress HER2. We analysed if the axis is activated in BCSC opening the door to a treatment that specifically targets this population.
2. Altered protein glycosylation
BCSC show alterations in the glycosylation patterns of their proteins. The activity of some of the enzymes that modulate protein glycosylation is necessary to maintain the stem compartment of the breast tumours. We have generated the first CRISPR library against every protein glycosylation gene annotated. This is an important tool that could be of the interest of multiple scientists worldwide and we will use it to determine which of these enzymes are essential for BCSC to survive.
The overall objective of the project is to identify new molecules in BCSC that can be targeted therapeutically, with the final goal of increasing the survival of breast cancer patients and improving their quality of life.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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 identificat

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The results obtained during this year link HER2 overexpression to the breast cancer stem cell population. Moreover, we have shown that the activation of the axis HER2-IL6-JAK2-STAT3-S100A8/9 is essential for these cells with stem characteristics to survive. Importantly, inhibitors for different levels of this pathway have been described and are used in the clinics for the treatment of different diseases. This is an opportunity to repurpose these drugs, and use them for the cure of breast cancer patients that do not respond to the current treatments.
We have also generated the first pooled CRISPRs library that targets all the protein glycosylation genes. The library could be of the interest of a large number of basic and translational scientist and its deposit at the repository will facilitate the access to any researcher interested on the library. At the same time, this will promote that the published paper describing the generation of the library becomes highly cited.
We will also deposit in Addgene repository the modified version of LentiCRIPR V2 containing the tGFP protein. A big challenge in setting up a CRISPR screening is the fact that the infection rate is not easily achieved. Our new version of the lentiviral vector will facilitate the screening protocol for researchers worldwide by allowing the calculation of the percentage of infected cells based on the fluorescence that they emit. This could be obtained using standard techniques as flow cytometry or fluorescent microscopy.

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