Periodic Reporting for period 1 - ROSETTA (Deciphering the Role of aberrant glycOSylation in the rEsponse to Targeted TherApies for breast cancer)
Reporting period: 2020-04-01 to 2022-03-31
Resistance and cancer progression remain a major obstacle to the successful treatment of cancer. In HER2-positive breast cancer, the development of targeted therapies, such as trastuzumab, has revolutionized the treatment for these cancer patients. However, a significant number of patients develop resistance. Therefore, understanding the causes of resistance is key for the development of better treatments. Multiple mechanisms contributing to the resistance to trastuzumab have been described. However, an important aspect of cancer cells has been largely overlooked: the aberrant glycosylation of cancer cells and how that could impact the efficacy of targeted therapies.
Therefore, the goal of this project was to provide a new focus to the challenge of resistance by deciphering the impact of glycosylation on resistance to HER2-targeted therapies. Aberrant glycosylation of proteins, including acquisition of unconventional N-glycosylation sites, is a hallmark in cancer and has been linked to multiple processes such as invasion of other tissues by tumor cells, tumor-induced angiogenesis, and immune response modulation. The receptor HER2, the main driver of HER2-positive breast cancers, is heavily glycosylated and is also the target of current antibody-based treatments, including trastuzumab. In this context, this project addressed (1) the identification of glycosylation-related genes associated with resistance to trastuzumab in patients to (2) dissect their role on trastuzumab binding and (3) assess their impact in the response to trastuzumab mediated by immune cells. This research has combined the analysis of clinical data from tumor biopsies with the use of trastuzumab-resistant tumor-derived breast cancer cell lines and implementation and imaging of 3D heterotypic cultures of cancer cell line-derived spheroids that also contain immune cells. Altogether, this project addressed a largely unexplored layer of complexity in cancer biology and, holds the potential to identify new markers of response to therapies and open the path to new therapeutic options for the improvement of current treatments.
Therefore, the goal of this project was to provide a new focus to the challenge of resistance by deciphering the impact of glycosylation on resistance to HER2-targeted therapies. Aberrant glycosylation of proteins, including acquisition of unconventional N-glycosylation sites, is a hallmark in cancer and has been linked to multiple processes such as invasion of other tissues by tumor cells, tumor-induced angiogenesis, and immune response modulation. The receptor HER2, the main driver of HER2-positive breast cancers, is heavily glycosylated and is also the target of current antibody-based treatments, including trastuzumab. In this context, this project addressed (1) the identification of glycosylation-related genes associated with resistance to trastuzumab in patients to (2) dissect their role on trastuzumab binding and (3) assess their impact in the response to trastuzumab mediated by immune cells. This research has combined the analysis of clinical data from tumor biopsies with the use of trastuzumab-resistant tumor-derived breast cancer cell lines and implementation and imaging of 3D heterotypic cultures of cancer cell line-derived spheroids that also contain immune cells. Altogether, this project addressed a largely unexplored layer of complexity in cancer biology and, holds the potential to identify new markers of response to therapies and open the path to new therapeutic options for the improvement of current treatments.
In order to identify clinically relevant glycogenes associated with the response to trastuzumab, we analyzed the expression of a large group of glycogenes in HER2-amplified breast cancer patients that received trastuzumab treatment. This analysis was performed in collaboration with Dr. van’t veer lab
at the University of California San Francisco and revealed 2 subsets of genes associated either with resistance or sensitivity to trastuzumab. Next, we addressed whether the overexpression or knock-down of the identified glycogenes affects the response of cancer cells to trastuzumab in vitro. In order to test this, we generated a panel of cancer cell models with a range of sensitivity to trastuzumab and our preliminary results suggests that reducing the expression of one of the identified glycogenes sensitizes resistant cancer cells to trastuzumab. Furthermore, a comprehensive analysis of the expression profiles of resistant and sensitive tumors using RNA sequencing, revealed that some of the glycogenes identified in patients were also potentially conferring resistance to trastuzumab in vivo using a cell line-derived xenograft mouse model.
The main mechanisms of action of trastuzumab requires the engagement of cells of the immune system. To elucidate whether the selected glycogenes limit an effective immune response towards tumor cells we designed and implemented a co-culture in vitro system combining engineered immune cells with HER2-amplified cancer cells. Our co-culture model shows specific cytotoxicity for HER2-amplified cells in the presence of trastuzumab and levels of cytotoxicity similar to immune cells isolated from donors. Thus, this co-culture model represents a valuable tool to study the impact of the identified glycogenes on the immune response mediated by trastuzumab. Furthermore, we have adapted this co-culture system to a 3D setting optimizing the automated generation and live-imaging of 3D cancer spheroids using advanced microscopy.
Overview and dissemination of results
• We identified a set of glycogenes associated with resistance to trastuzumab in patients and implemented the use of advanced co-culture systems and live-microscopy to mimic and visualize in vitro the impact of these glycogenes in the immune response induced by trastuzumab.
• RNA-sequencing of trastuzumab resistant and sensitive tumors have revealed new potential mechanism of resistance to trastuzumab.
• Results have been communicated to collaborators and local stakeholders in regular internal meetings and workshops. Results will be communicated to the scientific community through publication in scientific journals.
• Results have provided new teaching materials for M.Sc students in Molecular Medicine
• Results have been communicated to a scientific audience through oral presentations at (inter)national conferences and meetings.
at the University of California San Francisco and revealed 2 subsets of genes associated either with resistance or sensitivity to trastuzumab. Next, we addressed whether the overexpression or knock-down of the identified glycogenes affects the response of cancer cells to trastuzumab in vitro. In order to test this, we generated a panel of cancer cell models with a range of sensitivity to trastuzumab and our preliminary results suggests that reducing the expression of one of the identified glycogenes sensitizes resistant cancer cells to trastuzumab. Furthermore, a comprehensive analysis of the expression profiles of resistant and sensitive tumors using RNA sequencing, revealed that some of the glycogenes identified in patients were also potentially conferring resistance to trastuzumab in vivo using a cell line-derived xenograft mouse model.
The main mechanisms of action of trastuzumab requires the engagement of cells of the immune system. To elucidate whether the selected glycogenes limit an effective immune response towards tumor cells we designed and implemented a co-culture in vitro system combining engineered immune cells with HER2-amplified cancer cells. Our co-culture model shows specific cytotoxicity for HER2-amplified cells in the presence of trastuzumab and levels of cytotoxicity similar to immune cells isolated from donors. Thus, this co-culture model represents a valuable tool to study the impact of the identified glycogenes on the immune response mediated by trastuzumab. Furthermore, we have adapted this co-culture system to a 3D setting optimizing the automated generation and live-imaging of 3D cancer spheroids using advanced microscopy.
Overview and dissemination of results
• We identified a set of glycogenes associated with resistance to trastuzumab in patients and implemented the use of advanced co-culture systems and live-microscopy to mimic and visualize in vitro the impact of these glycogenes in the immune response induced by trastuzumab.
• RNA-sequencing of trastuzumab resistant and sensitive tumors have revealed new potential mechanism of resistance to trastuzumab.
• Results have been communicated to collaborators and local stakeholders in regular internal meetings and workshops. Results will be communicated to the scientific community through publication in scientific journals.
• Results have provided new teaching materials for M.Sc students in Molecular Medicine
• Results have been communicated to a scientific audience through oral presentations at (inter)national conferences and meetings.
We identified that specific glycogenes are associated with resistance to the HER2-targeting drug trastuzumab in HER2-amplified breast cancer patients. We are validating this finding in several cell models generated in vitro and vivo using multiple approaches, including the development of a relevant co-culture system. This co-culture model provides a valuable tool to study the interplay of immune and cancer cells and address the impact of mechanisms of resistance to trastuzumab mimicking in vitro the immune response elicited by trastuzumab in vivo.
The work initiated with this project holds the potential to reveal not only new biomarkers for response to HER2-targeted therapies, but also open new directions in the development of new and more specific therapies to improve future treatments based on of glycan-specific antibodies and/or small molecules for specific glycosyltransferases upregulated in cancer. Furthermore, the impact of this project extends beyond the field of breast cancer. Aberrant glycosylation may provide a novel and unique opportunity for clinical intervention and not only in breast cancer. Aberrant glycosylation has been observed in multiple cancer types and HER2-targeting therapies like trastuzumab are currently being used for the treatment of gastric, lung, colorectal and other cancer types.
The work initiated with this project holds the potential to reveal not only new biomarkers for response to HER2-targeted therapies, but also open new directions in the development of new and more specific therapies to improve future treatments based on of glycan-specific antibodies and/or small molecules for specific glycosyltransferases upregulated in cancer. Furthermore, the impact of this project extends beyond the field of breast cancer. Aberrant glycosylation may provide a novel and unique opportunity for clinical intervention and not only in breast cancer. Aberrant glycosylation has been observed in multiple cancer types and HER2-targeting therapies like trastuzumab are currently being used for the treatment of gastric, lung, colorectal and other cancer types.