Molecular & Cellular Oncology
ISSN: (Print) 2372-3556 (Online) Journal homepage: http://www.tandfonline.com/loi/kmco20
Co-targeting Cancer Stem-like Cells and Bulk Cancer Cells with a Bispecific Antibody Shi Hu To cite this article: Shi Hu (2017): Co-targeting Cancer Stem-like Cells and Bulk Cancer Cells with a Bispecific Antibody, Molecular & Cellular Oncology, DOI: 10.1080/23723556.2017.1308851 To link to this article: http://dx.doi.org/10.1080/23723556.2017.1308851
Accepted author version posted online: 28 Mar 2017.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kmco20 Download by: [University of Newcastle, Australia]
Date: 28 March 2017, At: 21:16
Author’s View Co-targeting Cancer Stem-like Cells and Bulk Cancer Cells with a Bispecific Antibody Shi Hu1,* 1
Department of Biophysics, College of Basic Medical Sciences, Second Military Medical University, Shanghai
200433, China *
To whom correspondence should be addressed: S.H ([email protected]; [email protected])
Abstract Epidermal growth factor receptor (EGFR) is a widely recognized target for tumors, but resistance is commonly reported. Recently, we reported that dual targeting of EGFR and NOTCH2/3 receptors with antibody CT16 showed a strong anti-stem effect both in vitro and in vivo to overcome resistance of EGFR inhibitors and radiation.
1
More than 90% of cancer related deaths are caused by tumor metastasis1. The metastasis formation can be considered as a culmination of the Darwinian evolutionary process within the tumor, when competing of multiple sub-clones results in the development of the cell inherent traits that favor tumor dissemination. The core of tumor evolution is cancer stem cell (CSC), which is defined by the self-renewal potential, clonogenic properties and genomic instability. The cancer cells which have the capacity to colonize distant organs have the features of CSC but also exert their tumor-initiating capacity under adverse micro-environmental conditions2. Therefore, the CSCs has received attention recent years.
CSCs are relatively resistant to cytotoxic chemotherapy, both in preclinical models and in neoadjuvant clinical trials3. Although both chemotherapy and radiation therapy may cause a notable loss of the bulk cancer cell population, these treatments actually stimulate CSC self-renewal through cytokine production and DNA repair mechanisms4. Additionally, current individualized treatments [such as Epidermal Growth Factor Receptor (EGFR) inhibitors] are mainly according to the molecular phenotype of the bulk cancer cell population, therefore show little effect on the CSC population. Tumors eventually become resistant to these drugs. In recent clinical trials, EGFR inhibitors combined with chemo-radiation had shown disappointing results. In the Radiation Therapy Oncology Group phase III trial (RTOG 0522), adding cetuximab to radiation and cisplatin treatment for patients with locally advanced head and neck cancer did not improve outcomes 5.
We focus on resistant mechanism of EGFR inhibitors and development of novel targeted agents to overcome the treatment resistance6,7. In a recent research8, we created a dual targeting antibody called CT16, which can recognize both the EGFR and NOTCH, simultaneously inhibiting both pathways. The antibody were also tested in mouse models of non–small cell lung cancer (NSCLC), and our result showed that it inhibited cancer stem cells and was more
2
effective than antibodies against the individual pathways alone and in combination, particularly in the presence of radiation treatment.
Mechanically, the dual targeting antibody CT16 adds CSC-targeting capacity to molecular subtype-specific therapies, achieves targeting both CSCs and bulk tumor cells, therefore enhances the therapeutic response (Fig. 1). Moreover, with the anti-EGFR arm recognizing EGFR in bulk cell subpopulations and the anti-NOTCH arm recognizing the surface receptor of CSCs, CT16 may passively recruit EGFR-positive bulk cells to CSCs and inhibit both EGFR and NOTCH signaling at both side. These effects could only be achieved by CT16, but not individual antibodies or simply combined the antibodies.
Our data suggested that dual targeting of EGFR and NOTCH signaling might prevent or delay acquired resistance to EGFR inhibitors, but not overcome established acquired resistance. In our experiments, for the EGFR blockade– nonresponsive cells, treatment with EGFR inhibitors resulted in no increase in the relative proportion of CSCs. Our results also showed that radiation upregulated the epithelial-mesenchymal transition (EMT) gene sets in tumor. EGFR blockade in combination with radiation strongly enhanced this effect, mainly via enrichment of high mesenchymal marker expressing cell subpopulations under treatment selection. EMT gene expression was heterogeneously regulated not only in different cell lines but also in different cell subpopulations. Additionally, DNA repair protein expression induced by radiation was only slightly reduced upon EGFR blockade in our in vivo results. Combination with tarextumab treatment dramatically reduced the increase in expression of radiation/EGFR blockade-induced EMT and DNA repair genes, and CT16 nearly abolished this effect compared to the control group through its anti-stem cell effect.
To our knowledge, no dual-targeting antibodies that inhibit both tumor bulk cells and CSCs has been reported previously. However, targeting both EGFR and NOTCH receptors, key regulators in bulk tumor cells 9 or CSCs10 3
respectively, with a single antibody not only blocked tumor growth in both cancer cell line models and patient derived xenografts (PDX) models by inhibiting both signalings, but also markedly abolished treatment induced CSC expansion. Theoretically, CT16 treatment will not have benefits for the non-responsive cells treated with EGFR inhibitors or radiation (no treatment induced CSC expansion), consistent with our observation that CT16 was not beneficial in treating cetuximab- or erlotinib-resistant cell lines. These data also indicates a complex cellular heterogeneity and plasticity in the development and outcome of resistance to EGFR blockade and radiation.
Although we provide evidence that targeting CSC with CT16 can be achieved, our in vivo efficacy models may not fully recapitulate human NSCLC, and the data was from a small number of animals. Moreover, the mechanisms responsible for these therapeutic effects of antibodies against CSC are currently not well characterized. We are now working on these issues. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Funding
Supported by the National Natural Science Foundation of China Grant (Grant No: 81602690), General Financial Grant from the China Postdoctoral Science Foundation (Grant No: 2016M593006) and Postdoctoral scientific research funds of Second Military Medical University.
Data and materials availability
requests for data and materials should be addressed to S.H ([email protected]; [email protected]).
4
1.
Mehlen P, Puisieux A. Metastasis: a question of life or death. Nature Reviews Cancer 2006;6(6):449-458.
2.
Peitzsch C, Tyutyunnykova A, Pantel K, Dubrovska A. Cancer stem cells: the root of tumor recurrence and metastasis. Semin Cancer Biol 2017.
3.
Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, Pratesi G, Fabbri A, Andriani F, Tinelli S and others. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci U S A 2009;106(38):16281-6.
4.
Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu M-F, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. Journal of the National Cancer Institute 2008;100(9):672-679.
5.
Ang KK, Zhang Q, Rosenthal DI, Nguyen-Tan PF, Sherman EJ, Weber RS, Galvin JM, Bonner JA, Harris J, El-Naggar AK. Randomized phase III trial of concurrent accelerated radiation plus cisplatin with or without cetuximab for stage III to IV head and neck carcinoma: RTOG 0522. Journal of Clinical Oncology 2014:JCO. 2013.53. 5633.
6.
Hu S, Dai H, Li T, Tang Y, Fu W, Yuan Q, Wang F, Lv G, Lv Y, Fan X and others. Broad RTK-targeted therapy overcomes molecular heterogeneity-driven resistance to cetuximab via vectored immunoprophylaxis in colorectal cancer. Cancer Lett 2016;382(1):32-43.
7.
Hu S, Fu W, Xu W, Yang Y, Cruz M, Berezov SD, Jorissen D, Takeda H, Zhu W. Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk. Cancer Res 2015;75(1):159-70.
5
8.
Hu S, Fu W, Li T, Yuan Q, Wang F, Lv G, Lv Y, Fan X, Shen Y, Lin F. Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency. Science Translational Medicine 2017;9(380):eaag0339.
9.
Arteaga CL, Engelman JA. ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer cell 2014;25(3):282-303.
10.
Takebe N, Nguyen D, Yang SX. Targeting notch signaling pathway in cancer: clinical development advances and challenges. Pharmacology & therapeutics 2014;141(2):140-149.
6
Figure 1. Co-targeting Cancer stem-like cells and bulk cancer cells. According to the cancer stem cell (CSC) model, epidermal growth factor receptor (EGFR) blockage and radiotherapy kills bulk tumor cells but leaves rare, CSCs untouched (up). These cells then drive cancer relapse and resistance. Stem cell targeting therapy may have little effect on the bulk cells, lead to tumor regression over time (center), but co-targeting CSCs and bulk cancer cells could be most effective, especially combined with radiotherapy (down).
7
ISSN: (Print) 2372-3556 (Online) Journal homepage: http://www.tandfonline.com/loi/kmco20
Co-targeting Cancer Stem-like Cells and Bulk Cancer Cells with a Bispecific Antibody Shi Hu To cite this article: Shi Hu (2017): Co-targeting Cancer Stem-like Cells and Bulk Cancer Cells with a Bispecific Antibody, Molecular & Cellular Oncology, DOI: 10.1080/23723556.2017.1308851 To link to this article: http://dx.doi.org/10.1080/23723556.2017.1308851
Accepted author version posted online: 28 Mar 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kmco20 Download by: [University of Newcastle, Australia]
Date: 28 March 2017, At: 21:16
Author’s View Co-targeting Cancer Stem-like Cells and Bulk Cancer Cells with a Bispecific Antibody Shi Hu1,* 1
Department of Biophysics, College of Basic Medical Sciences, Second Military Medical University, Shanghai
200433, China *
To whom correspondence should be addressed: S.H ([email protected]; [email protected])
Abstract Epidermal growth factor receptor (EGFR) is a widely recognized target for tumors, but resistance is commonly reported. Recently, we reported that dual targeting of EGFR and NOTCH2/3 receptors with antibody CT16 showed a strong anti-stem effect both in vitro and in vivo to overcome resistance of EGFR inhibitors and radiation.
1
More than 90% of cancer related deaths are caused by tumor metastasis1. The metastasis formation can be considered as a culmination of the Darwinian evolutionary process within the tumor, when competing of multiple sub-clones results in the development of the cell inherent traits that favor tumor dissemination. The core of tumor evolution is cancer stem cell (CSC), which is defined by the self-renewal potential, clonogenic properties and genomic instability. The cancer cells which have the capacity to colonize distant organs have the features of CSC but also exert their tumor-initiating capacity under adverse micro-environmental conditions2. Therefore, the CSCs has received attention recent years.
CSCs are relatively resistant to cytotoxic chemotherapy, both in preclinical models and in neoadjuvant clinical trials3. Although both chemotherapy and radiation therapy may cause a notable loss of the bulk cancer cell population, these treatments actually stimulate CSC self-renewal through cytokine production and DNA repair mechanisms4. Additionally, current individualized treatments [such as Epidermal Growth Factor Receptor (EGFR) inhibitors] are mainly according to the molecular phenotype of the bulk cancer cell population, therefore show little effect on the CSC population. Tumors eventually become resistant to these drugs. In recent clinical trials, EGFR inhibitors combined with chemo-radiation had shown disappointing results. In the Radiation Therapy Oncology Group phase III trial (RTOG 0522), adding cetuximab to radiation and cisplatin treatment for patients with locally advanced head and neck cancer did not improve outcomes 5.
We focus on resistant mechanism of EGFR inhibitors and development of novel targeted agents to overcome the treatment resistance6,7. In a recent research8, we created a dual targeting antibody called CT16, which can recognize both the EGFR and NOTCH, simultaneously inhibiting both pathways. The antibody were also tested in mouse models of non–small cell lung cancer (NSCLC), and our result showed that it inhibited cancer stem cells and was more
2
effective than antibodies against the individual pathways alone and in combination, particularly in the presence of radiation treatment.
Mechanically, the dual targeting antibody CT16 adds CSC-targeting capacity to molecular subtype-specific therapies, achieves targeting both CSCs and bulk tumor cells, therefore enhances the therapeutic response (Fig. 1). Moreover, with the anti-EGFR arm recognizing EGFR in bulk cell subpopulations and the anti-NOTCH arm recognizing the surface receptor of CSCs, CT16 may passively recruit EGFR-positive bulk cells to CSCs and inhibit both EGFR and NOTCH signaling at both side. These effects could only be achieved by CT16, but not individual antibodies or simply combined the antibodies.
Our data suggested that dual targeting of EGFR and NOTCH signaling might prevent or delay acquired resistance to EGFR inhibitors, but not overcome established acquired resistance. In our experiments, for the EGFR blockade– nonresponsive cells, treatment with EGFR inhibitors resulted in no increase in the relative proportion of CSCs. Our results also showed that radiation upregulated the epithelial-mesenchymal transition (EMT) gene sets in tumor. EGFR blockade in combination with radiation strongly enhanced this effect, mainly via enrichment of high mesenchymal marker expressing cell subpopulations under treatment selection. EMT gene expression was heterogeneously regulated not only in different cell lines but also in different cell subpopulations. Additionally, DNA repair protein expression induced by radiation was only slightly reduced upon EGFR blockade in our in vivo results. Combination with tarextumab treatment dramatically reduced the increase in expression of radiation/EGFR blockade-induced EMT and DNA repair genes, and CT16 nearly abolished this effect compared to the control group through its anti-stem cell effect.
To our knowledge, no dual-targeting antibodies that inhibit both tumor bulk cells and CSCs has been reported previously. However, targeting both EGFR and NOTCH receptors, key regulators in bulk tumor cells 9 or CSCs10 3
respectively, with a single antibody not only blocked tumor growth in both cancer cell line models and patient derived xenografts (PDX) models by inhibiting both signalings, but also markedly abolished treatment induced CSC expansion. Theoretically, CT16 treatment will not have benefits for the non-responsive cells treated with EGFR inhibitors or radiation (no treatment induced CSC expansion), consistent with our observation that CT16 was not beneficial in treating cetuximab- or erlotinib-resistant cell lines. These data also indicates a complex cellular heterogeneity and plasticity in the development and outcome of resistance to EGFR blockade and radiation.
Although we provide evidence that targeting CSC with CT16 can be achieved, our in vivo efficacy models may not fully recapitulate human NSCLC, and the data was from a small number of animals. Moreover, the mechanisms responsible for these therapeutic effects of antibodies against CSC are currently not well characterized. We are now working on these issues. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Funding
Supported by the National Natural Science Foundation of China Grant (Grant No: 81602690), General Financial Grant from the China Postdoctoral Science Foundation (Grant No: 2016M593006) and Postdoctoral scientific research funds of Second Military Medical University.
Data and materials availability
requests for data and materials should be addressed to S.H ([email protected]; [email protected]).
4
1.
Mehlen P, Puisieux A. Metastasis: a question of life or death. Nature Reviews Cancer 2006;6(6):449-458.
2.
Peitzsch C, Tyutyunnykova A, Pantel K, Dubrovska A. Cancer stem cells: the root of tumor recurrence and metastasis. Semin Cancer Biol 2017.
3.
Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, Pratesi G, Fabbri A, Andriani F, Tinelli S and others. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci U S A 2009;106(38):16281-6.
4.
Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu M-F, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. Journal of the National Cancer Institute 2008;100(9):672-679.
5.
Ang KK, Zhang Q, Rosenthal DI, Nguyen-Tan PF, Sherman EJ, Weber RS, Galvin JM, Bonner JA, Harris J, El-Naggar AK. Randomized phase III trial of concurrent accelerated radiation plus cisplatin with or without cetuximab for stage III to IV head and neck carcinoma: RTOG 0522. Journal of Clinical Oncology 2014:JCO. 2013.53. 5633.
6.
Hu S, Dai H, Li T, Tang Y, Fu W, Yuan Q, Wang F, Lv G, Lv Y, Fan X and others. Broad RTK-targeted therapy overcomes molecular heterogeneity-driven resistance to cetuximab via vectored immunoprophylaxis in colorectal cancer. Cancer Lett 2016;382(1):32-43.
7.
Hu S, Fu W, Xu W, Yang Y, Cruz M, Berezov SD, Jorissen D, Takeda H, Zhu W. Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk. Cancer Res 2015;75(1):159-70.
5
8.
Hu S, Fu W, Li T, Yuan Q, Wang F, Lv G, Lv Y, Fan X, Shen Y, Lin F. Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency. Science Translational Medicine 2017;9(380):eaag0339.
9.
Arteaga CL, Engelman JA. ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics. Cancer cell 2014;25(3):282-303.
10.
Takebe N, Nguyen D, Yang SX. Targeting notch signaling pathway in cancer: clinical development advances and challenges. Pharmacology & therapeutics 2014;141(2):140-149.
6
Figure 1. Co-targeting Cancer stem-like cells and bulk cancer cells. According to the cancer stem cell (CSC) model, epidermal growth factor receptor (EGFR) blockage and radiotherapy kills bulk tumor cells but leaves rare, CSCs untouched (up). These cells then drive cancer relapse and resistance. Stem cell targeting therapy may have little effect on the bulk cells, lead to tumor regression over time (center), but co-targeting CSCs and bulk cancer cells could be most effective, especially combined with radiotherapy (down).
7
