Final Report Summary - SHP2N (Characterization of nuclear SHP2 function in breast cancer progression and metastasis.)
• An executive summary (not exceeding 1 page).
This project was established to investigate the role of a protein tyrosine phosphatase (PTP), Src-homology 2 domain-containing phosphatase (SHP2), in breast cancer progression and metastasis. Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) control a reversible phosphorylation of tyrosine residues on protein substrates that can impact essentially all signaling pathways in the cell. Abnormal changes in tyrosine phosphorylation can affect cell fate and growth among other cellular functions and, therefore, are intimately linked with a broad spectrum of diseases, including breast cancer. Recent studies have clearly shown an active and critical role for PTPs in potentiating and/or sustaining cellular signals and their function in breast cancer are beginning to be elucidated. Therefore, the previously accepted notion that PTPs function as merely passive, house-keeping members of this “duo” is now being reconsidered and, for example, development of SHP2 inhibitors are currently underway.
This project began in 2015 with two main objectives: 1) Determine the nuclear function of SHP2 in mammary epithelial and breast cancer progression and metastasis and 2) Validate nucleolin (NCL) as a potential SHP2 nuclear substrate and assess whether it is a critical downstream effector of SHP2 in breast cancer.
Although still in the beginning stages of the project, it can be concluded that SHP2 is both a nuclear and cytosolic protein in human breast cancer and normal mammary epithelial cells. To better investigate this observation in more detail, novel tools (lenti-viral vector construction of mutant SHP2 of a putative nuclear export signal) and cell lines (SHP2 null and mutant cells lines using CRISPR-CAS9 technology) were developed. Indeed, SHP2 and NCL are nuclear interactors within the cell populations mentioned above. Although NCL does not appear to be a protein dependent on SHP2’s PTP activity, it could be hypothesized that these two molecules act together to promote other PTP-independent functions within the nucleus.
This proposal is the first to examine the nuclear- and perhaps epigenetic- function of SHP2 in breast cancer. When pursued further, these studies will surely help to better understand the mechanistic role that SHP2 has in not only human breast cancer but also in other solid tumors. For example, if one function of SHP2, either cytosolic or nuclear, is dominant over the other. They will also identify which function, if different, is pro- or inhibitory for breast cancer progression and/or metastasis. The implication of this work suggests avenues/pathways for novel therapeutic targeting of SHP2 activity in breast cancer.
• A summary description of project context and objectives (not exceeding 4 pages).
Breast cancer is the most frequently diagnosed cancer in women with ~1.1 million worldwide cases (www.uicc.org (1). Despite intense study, in a significant fraction of women, tumor progression and disease relapse is inevitable underscoring a crucial gap in our understanding of these important hallmarks. Although a large research effort has been made, our current understanding of the biological mechanisms driving breast tumorigenesis, progression and disease relapse is still limited. To date little is known about the biochemical networks controlling this critical hallmark (2). We have previously identified a role for the protein tyrosine phosphatase (PTP), Src-homology 2 domain-containing phosphatase (SHP2), in the maintenance and progression of HER2+ and triple negative breast cancer, both subtypes conferring a dismal prognosis (3). Our data revealed a nuclear localization for SHP2 but the effects of nuclear SHP2 remain unknown. Moreover, our preliminary data using an unbiased proteomic analysis of nuclear extracts from breast cancer cells revealed potential SHP2 substrates including nucleolin. This proposal aimed to: 1) Determine the nuclear function of SHP2 in mammary epithelial cells and breast cancer progression and metastasis, and 2) Validate nucleolin (NCL) as a potential SHP2 nuclear substrate and assess whether it is a critical downstream effector of SHP2 in breast cancer.
• A description of the main S&T results/foregrounds (not exceeding 25 pages),
The main results obtained for the SHP2N proposal are outlined below:
Objective 1): Determine the nuclear function of SHP2 in mammary epithelial cells and breast cancer progression and metastasis. Preliminary data from our lab demonstrated that SHP2 is present in the nucleus of human breast cancer cell lines. In 2015, I was able to validate our previous finding that SHP2 can be found in the nucleus of a human breast cancer cell line and a non-tumorigenic but transformed human cell line (SUM159 and MCF10AHER2/3, respectively). Immunoblotting of lysates derived from nuclear and cytoplasmic fractions revealed the presence of SHP2 in both the cytoplasmic and nuclear fractions. However, efficient knockdown of SHP2 was not obtained using a lentiviral vector expressing targeted shRNAs. Therefore, we utilized CRISPR technology to generate data where more certain conclusions could be made allowing us to better address this aim. Thus, I developed and carried out experiments to generate SHP2 cell lines expressing mutations in the nuclear localization sequence (NLS, as described in my original proposal) using homologous recombination. As a back-up strategy, I also developed SHP2 null lines that would allow for exogenous expression of lenti-viral vectors expressing the SHP2 NLS and NES (see below) mutants. Finally, as the NLS mutant only sequesters SHP2 in the cytosol, we identified a putative nuclear export sequence (NES) in SHP2 using the NETNES1.1 predictive server (http://www.cbs.dtu.dk/services/NetNES/). I generated NES mutants in the lenti-viral system used to generate the NLS mutants and have performed preliminary experiments to validate the SHP2 NES in human breast cancer cell lines. Ideally, this tool would enable us to sequester SHP2 in the nucleus and better address any putative nuclear function of SHP2. These newly generated tools, when used together, should enable us to accurately address if nuclear SHP2 expression has effects on breast cancer progression and metastasis in vitro and in vivo.
Objective 2): Assess the putative effects of nucleolin, as an SHP2 effector, in the nucleus of mammary epithelial and breast cancer cells. Given that nuclear SHP2 is observed in breast cancer cell lines, the identification of its downstream effectors is of paramount interest to understand the oncogenic effects of SHP2. To address this point, our lab employed an unbiased, proteomic approach to identify SHP2 interacting proteins by performing quantitative stable isotope labeling by amino acids in cell culture (SILAC) using a catalytically dead SHP2 double trapping mutant (SHP2 D425A+C459S). Wild-type- and trapping mutant- SHP2 proteins were expressed in SUM159 breast cancer cells and subsequently immunoprecipitated from cytosolic and nuclear protein fractions. Results from these SILAC experiments identified NCL as a novel SHP2 interacting protein in the nuclear fraction. In 2015, I was able to validate that nucleolin is indeed a SHP2 nuclear substrate through co-immunoprecipitation experiments of SHP2 and NCL from nuclear lysates in breast cancer- and normal mammary epithelial- cell lines (SUM159 and MCF10A, respectively). In addition, I also examined if NCL shows increased tyrosyl phosphorylation in cells expressing shRNA miR against SHP2. However, immunoblotting did not reveal any difference in the levels of tyrosine phosphorylation on the NCL protein when comparing WT and SHP2 knock down SUM159 or MCF10A cell lines. These data would suggest that the effects resulting from the nuclear interaction between SHP2 and NCL is not dependant on tyrosine phosphorylation. Currently, we are exploring an epigenetic mechanism of nuclear SHP2 and if this is impacted by its interaction with NCL.
• The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
It is becoming clear that PTPs are no longer considered to be only “house-keepers” of cellular homeostasis but rather play an active role in controlling cell fate, transformation and tumor progression. Our experiments in 2015 have generated new tools that will surely address several outstanding questions regarding to role of SHP2 in breast cancer progression and metastasis. Moreover, they will hopefully shed light onto a complex role that SHP2 possesses both at the tumoral level (progression and metastasis) and the molecular level (nucleus vs cytoplasmic expression). Currently, studies on SHP2 published in 2014-2015 are continually demonstrating it’s key role in breast cancer biology at the cellular level. To my knowledge, our study is the first to examine the nuclear- and perhaps epigenetic- function of SHP2 in breast cancer. If confirmed, it would identify a new avenue for therapeutic targeting of SHP2 activity in breast cancer. I expect these findings could open a new area of SHP2 biology that will be pursued by the breast cancer community and beyond.
• The address of the project public website, if applicable as well as relevant contact details.
N/A
This project was established to investigate the role of a protein tyrosine phosphatase (PTP), Src-homology 2 domain-containing phosphatase (SHP2), in breast cancer progression and metastasis. Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) control a reversible phosphorylation of tyrosine residues on protein substrates that can impact essentially all signaling pathways in the cell. Abnormal changes in tyrosine phosphorylation can affect cell fate and growth among other cellular functions and, therefore, are intimately linked with a broad spectrum of diseases, including breast cancer. Recent studies have clearly shown an active and critical role for PTPs in potentiating and/or sustaining cellular signals and their function in breast cancer are beginning to be elucidated. Therefore, the previously accepted notion that PTPs function as merely passive, house-keeping members of this “duo” is now being reconsidered and, for example, development of SHP2 inhibitors are currently underway.
This project began in 2015 with two main objectives: 1) Determine the nuclear function of SHP2 in mammary epithelial and breast cancer progression and metastasis and 2) Validate nucleolin (NCL) as a potential SHP2 nuclear substrate and assess whether it is a critical downstream effector of SHP2 in breast cancer.
Although still in the beginning stages of the project, it can be concluded that SHP2 is both a nuclear and cytosolic protein in human breast cancer and normal mammary epithelial cells. To better investigate this observation in more detail, novel tools (lenti-viral vector construction of mutant SHP2 of a putative nuclear export signal) and cell lines (SHP2 null and mutant cells lines using CRISPR-CAS9 technology) were developed. Indeed, SHP2 and NCL are nuclear interactors within the cell populations mentioned above. Although NCL does not appear to be a protein dependent on SHP2’s PTP activity, it could be hypothesized that these two molecules act together to promote other PTP-independent functions within the nucleus.
This proposal is the first to examine the nuclear- and perhaps epigenetic- function of SHP2 in breast cancer. When pursued further, these studies will surely help to better understand the mechanistic role that SHP2 has in not only human breast cancer but also in other solid tumors. For example, if one function of SHP2, either cytosolic or nuclear, is dominant over the other. They will also identify which function, if different, is pro- or inhibitory for breast cancer progression and/or metastasis. The implication of this work suggests avenues/pathways for novel therapeutic targeting of SHP2 activity in breast cancer.
• A summary description of project context and objectives (not exceeding 4 pages).
Breast cancer is the most frequently diagnosed cancer in women with ~1.1 million worldwide cases (www.uicc.org (1). Despite intense study, in a significant fraction of women, tumor progression and disease relapse is inevitable underscoring a crucial gap in our understanding of these important hallmarks. Although a large research effort has been made, our current understanding of the biological mechanisms driving breast tumorigenesis, progression and disease relapse is still limited. To date little is known about the biochemical networks controlling this critical hallmark (2). We have previously identified a role for the protein tyrosine phosphatase (PTP), Src-homology 2 domain-containing phosphatase (SHP2), in the maintenance and progression of HER2+ and triple negative breast cancer, both subtypes conferring a dismal prognosis (3). Our data revealed a nuclear localization for SHP2 but the effects of nuclear SHP2 remain unknown. Moreover, our preliminary data using an unbiased proteomic analysis of nuclear extracts from breast cancer cells revealed potential SHP2 substrates including nucleolin. This proposal aimed to: 1) Determine the nuclear function of SHP2 in mammary epithelial cells and breast cancer progression and metastasis, and 2) Validate nucleolin (NCL) as a potential SHP2 nuclear substrate and assess whether it is a critical downstream effector of SHP2 in breast cancer.
• A description of the main S&T results/foregrounds (not exceeding 25 pages),
The main results obtained for the SHP2N proposal are outlined below:
Objective 1): Determine the nuclear function of SHP2 in mammary epithelial cells and breast cancer progression and metastasis. Preliminary data from our lab demonstrated that SHP2 is present in the nucleus of human breast cancer cell lines. In 2015, I was able to validate our previous finding that SHP2 can be found in the nucleus of a human breast cancer cell line and a non-tumorigenic but transformed human cell line (SUM159 and MCF10AHER2/3, respectively). Immunoblotting of lysates derived from nuclear and cytoplasmic fractions revealed the presence of SHP2 in both the cytoplasmic and nuclear fractions. However, efficient knockdown of SHP2 was not obtained using a lentiviral vector expressing targeted shRNAs. Therefore, we utilized CRISPR technology to generate data where more certain conclusions could be made allowing us to better address this aim. Thus, I developed and carried out experiments to generate SHP2 cell lines expressing mutations in the nuclear localization sequence (NLS, as described in my original proposal) using homologous recombination. As a back-up strategy, I also developed SHP2 null lines that would allow for exogenous expression of lenti-viral vectors expressing the SHP2 NLS and NES (see below) mutants. Finally, as the NLS mutant only sequesters SHP2 in the cytosol, we identified a putative nuclear export sequence (NES) in SHP2 using the NETNES1.1 predictive server (http://www.cbs.dtu.dk/services/NetNES/). I generated NES mutants in the lenti-viral system used to generate the NLS mutants and have performed preliminary experiments to validate the SHP2 NES in human breast cancer cell lines. Ideally, this tool would enable us to sequester SHP2 in the nucleus and better address any putative nuclear function of SHP2. These newly generated tools, when used together, should enable us to accurately address if nuclear SHP2 expression has effects on breast cancer progression and metastasis in vitro and in vivo.
Objective 2): Assess the putative effects of nucleolin, as an SHP2 effector, in the nucleus of mammary epithelial and breast cancer cells. Given that nuclear SHP2 is observed in breast cancer cell lines, the identification of its downstream effectors is of paramount interest to understand the oncogenic effects of SHP2. To address this point, our lab employed an unbiased, proteomic approach to identify SHP2 interacting proteins by performing quantitative stable isotope labeling by amino acids in cell culture (SILAC) using a catalytically dead SHP2 double trapping mutant (SHP2 D425A+C459S). Wild-type- and trapping mutant- SHP2 proteins were expressed in SUM159 breast cancer cells and subsequently immunoprecipitated from cytosolic and nuclear protein fractions. Results from these SILAC experiments identified NCL as a novel SHP2 interacting protein in the nuclear fraction. In 2015, I was able to validate that nucleolin is indeed a SHP2 nuclear substrate through co-immunoprecipitation experiments of SHP2 and NCL from nuclear lysates in breast cancer- and normal mammary epithelial- cell lines (SUM159 and MCF10A, respectively). In addition, I also examined if NCL shows increased tyrosyl phosphorylation in cells expressing shRNA miR against SHP2. However, immunoblotting did not reveal any difference in the levels of tyrosine phosphorylation on the NCL protein when comparing WT and SHP2 knock down SUM159 or MCF10A cell lines. These data would suggest that the effects resulting from the nuclear interaction between SHP2 and NCL is not dependant on tyrosine phosphorylation. Currently, we are exploring an epigenetic mechanism of nuclear SHP2 and if this is impacted by its interaction with NCL.
• The potential impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and exploitation of results (not exceeding 10 pages).
It is becoming clear that PTPs are no longer considered to be only “house-keepers” of cellular homeostasis but rather play an active role in controlling cell fate, transformation and tumor progression. Our experiments in 2015 have generated new tools that will surely address several outstanding questions regarding to role of SHP2 in breast cancer progression and metastasis. Moreover, they will hopefully shed light onto a complex role that SHP2 possesses both at the tumoral level (progression and metastasis) and the molecular level (nucleus vs cytoplasmic expression). Currently, studies on SHP2 published in 2014-2015 are continually demonstrating it’s key role in breast cancer biology at the cellular level. To my knowledge, our study is the first to examine the nuclear- and perhaps epigenetic- function of SHP2 in breast cancer. If confirmed, it would identify a new avenue for therapeutic targeting of SHP2 activity in breast cancer. I expect these findings could open a new area of SHP2 biology that will be pursued by the breast cancer community and beyond.
• The address of the project public website, if applicable as well as relevant contact details.
N/A