PDF file - 187K, Enhanced aggressiveness of C6 and HER2 cells

PDF file - 540K, Protein profiling analysis of HER2+ versus HER2- BCSCs

PDF file - 144K, FACS of irradiated MCF7 cells and xenograft tumors

PDF file - 36K, Mitochondrial membrane potential in HER2 siRNA treated cells

PDF file - 89K, Enhanced gap filling rates in HER2+ versus HER2- BCSCs

PDF file - 513K, IHS of co-activation of HER2/CD44 cells in tumor tissues

PDF file - 79K, Peptide and protein HPLC-MS/MS identification data for all proteins included in Table S1

PDF file - 80K, Proteins identified by HPLC-MS/MS analysis are categorized into functional groups

Although the efficacy of cancer radiotherapy (RT) can be enhanced by targeted immunotherapy, the immunosuppressive factors induced by radiation on tumor cells remain to be identified. Here, we report that CD47-mediated anti-phagocytosis is concurrently upregulated with HER2 in radioresistant breast cancer (BC) cells and RT-treated mouse syngeneic BC. Co-expression of both receptors is more frequently detected in recurrent BC patients with poor prognosis. CD47 is upregulated preferentially in HER2-expressing cells, and blocking CD47 or HER2 reduces both receptors with diminished clonogenicity and augmented phagocytosis. CRISPR-mediated CD47 and HER2 dual knockouts not only inhibit clonogenicity but also enhance macrophage-mediated attack. Dual antibody of both receptors synergizes with RT in control of syngeneic mouse breast tumor. These results provide the evidence that aggressive behavior of radioresistant BC is caused by CD47-mediated anti-phagocytosis conjugated with HER2-prompted proliferation. Dual blockade of CD47 and HER2 is suggested to eliminate resistant cancer cells in BC radiotherapy.

Accumulating studies are aimed to combine immunoregulation with the standard of treatment such as radiotherapy in anti-cancer therapy. A potential cross resistance of tumor cells will be a barrier in furthering the long-term control by such combined treatment. CD47, an immunoglobulin-like transmembrane protein, enables cancer cells to escape the immune surveillance by flagging them with a ‘don’t eat me’ signal, which is recognized by the signal regulatory protein α (SIRPα) on macrophages. Here, we report that CD47 expression is enhanced by radiation in breast cancer cells and in vivo irradiated xenograft tumors via NF-κB regulation. In addition, CD47 and HER2 are co-induced by radiation and CD47 expression is specially enhanced by radiation in HER2-expressing cells. Contrasted with primary tumors, co-expression of CD47 and HER2 is elevated in the recurrent breast tumors. Anti-HER2 treatment inhibits CD47 transcription and protein expression with increased macrophage phagocytosis. Likewise, anti-CD47 not only augments macrophage phagocytosis but also reduces the capacity of tumorsphere formation. Radiation with anti-CD47 synergistically suppresses clonogenic survival and the maximal inhibition was achieved by dual inhibition of CD47 and HER2 with radiation. The synergy of in vivo tumor inhibition is established in mouse breast tumor model via local tumor radiotherapy combined with anti-CD47 treatment. These results demonstrate that the HER2-NF-κB-CD47 loop is responsible the cross-resistance of breast tumor against immune surveillance and radiation. Thus dual inhibition of CD47 and HER2 is a potential target to enhance tumor response to immune-radiotherapy. Funding: This study was partially supported by National Cancer Institute RO1 grant R01CA213830-01 (JJL). Note: This abstract was not presented at the meeting. Citation Format: Demet Candas, Lu Zhang, Cheikh Menaa, Ming Fan, Yanhong Zhang, Lin Liu, Weibing Zhou, Soheila Azghadi, Colleen Sweeney, Rulong Shen, Tzu-yin Lin, Chong-xian Pan, Andrew T. Vaughan, Ji Ming Wang, Hong-Wu Chen, Kit Lam, Lun-Quan Sun, Arta M. Monjazeb, William J. Murphy, Jian Jian Li. Dual inhibition of CD47 and HER2 to radiosensitize breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-226. doi:10.1158/1538-7445.AM2017-LB-226

Granzyme A (GzmA) levels are elevated in the plasma and synovium of patients with rheumatoid arthritis (RA), suggesting involvement of this protease in the pathogenesis of the disease. GzmA contributes to sepsis by regulating the production of proinflammatory cytokines. The purpose of this study was to evaluate the contribution of GzmA to the pathogenesis of RA in vivo and to examine the possibility that GzmA acting via tumor necrosis factor (TNF) stimulates osteoclastogenesis.

Abstract Accumulating studies are aimed to combine immunoregulation with the standard of treatment such as radiotherapy in anti-cancer therapy. A potential cross resistance of tumor cells will be a barrier in furthering the long-term control by such combined treatment. CD47, an immunoglobulin-like transmembrane protein, enables cancer cells to escape the immune surveillance by flagging them with a ‘don’t eat me’ signal, which is recognized by the signal regulatory protein α (SIRPα) on macrophages. Here, we report that CD47 expression is enhanced by radiation in breast cancer cells and in vivo irradiated xenograft tumors via NF-κB regulation. In addition, CD47 and HER2 are co-induced by radiation and CD47 expression is specially enhanced by radiation in HER2-expressing cells. Contrasted with primary tumors, co-expression of CD47 and HER2 is elevated in the recurrent breast tumors. Anti-HER2 treatment inhibits CD47 transcription and protein expression with increased macrophage phagocytosis. Likewise, anti-CD47 not only augments macrophage phagocytosis but also reduces the capacity of tumorsphere formation. Radiation with anti-CD47 synergistically suppresses clonogenic survival and the maximal inhibition was achieved by dual inhibition of CD47 and HER2 with radiation. The synergy of in vivo tumor inhibition is established in mouse breast tumor model via local tumor radiotherapy combined with anti-CD47 treatment. These results demonstrate that the HER2-NF-κB-CD47 loop is responsible the cross-resistance of breast tumor against immune surveillance and radiation. Thus dual inhibition of CD47 and HER2 is a potential target to enhance tumor response to immune-radiotherapy. Funding: This study was partially supported by National Cancer Institute RO1 grant R01CA213830-01 (JJL). Note: This abstract was not presented at the meeting. Citation Format: Demet Candas, Lu Zhang, Cheikh Menaa, Ming Fan, Yanhong Zhang, Lin Liu, Weibing Zhou, Soheila Azghadi, Colleen Sweeney, Rulong Shen, Tzu-yin Lin, Chong-xian Pan, Andrew T. Vaughan, Ji Ming Wang, Hong-Wu Chen, Kit Lam, Lun-Quan Sun, Arta M. Monjazeb, William J. Murphy, Jian Jian Li. Dual inhibition of CD47 and HER2 to radiosensitize breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-226. doi:10.1158/1538-7445.AM2017-LB-226

Background: Circadian deregulation is associated with the development of numerous diseases, including cancer, diabetes, and several neurological disorders. A recent study shows that the onset of carcinogenicity is specifically linked to the time of exposures (day or night) to ultraviolet light and ionizing radiation in mice. This effect is believed to be mediated through radiation-induced DNA damage and deregulation of the WNT/β-Catenin signaling pathway, hence affecting cell survival and proliferation. Results: The current studies aim to elucidate the contribution of a core circadian gene, Period2 (Per2), in the radioprotective response of mice bone marrow. Epithelial cells and mice bone marrow express PER2 in contrast to cancer cells. Bone marrow isolated from Per2 knockout mice exhibit radioresistence to high doses of ionizing radiation (HDIR; 2Gy) compared to wild-type mice controls suggesting that Per2 expression was responsible for radiation-induced cell death. This finding was translated by increased granulocyte-macrophage (GM-CFUs) clonogenic survival and reduced cell apoptosis. Mechanistic analysis showed that Per2 knockout mice bone marrow expressed higher levels of procaspase3 and less expression of active caspase3 after HDIR treatment compared to wild-type mice bone marrow and sham irradiated controls. Furthermore, Per2 knockout mice treated with 12Gy total body radiation (TBIR) showed an 80% survival verses 30% survival in wild-type mice. Whole genome-wide sequencing detected signal transduction pathways reflecting enhanced DNA damage repair capacity in knockout mice bone marrow, hence defining the role of PER2 in the radioresistant phenotype in vivo. Conclusions: This data is the first evidence supporting the critical role of PER2 in protecting mice hematopoietic stem cells (HSCs) from HDIR via enhancement of genes involved in DNA repair signaling networks. Citation Format: Aris Alexandrou, Cheikh Menaa, Chris Liu, Steven Pai, Kai Xiao, Ming Fan, Shuaib Juma, Loning Fu, William J. Murphy, Jian Jian Li. The role of PERIOD2 for radioprotection against ionizing radiation in mice bone marrow. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5107. doi:10.1158/1538-7445.AM2014-5107

Abstract The tyrosine kinase receptor HER2 (ErbB2/neu) is predominantly localized to the basolateral membrane of the epithelium and has been implicated in mammary tumorigenesis as well as in resistance to chemotherapy and radiation. Here we demonstrate that HER2 interacts with discs large 1 (DLG1) that is one of PDZ domain proteins expressed at the basolateral membrane of human breast epithelial cells and plays an important role in the establishment and maintenance of epithelial polarity in Drosophila. However, either loss of the last 6 C-terminal amino acids (HER2-Δ1250-1255) or a valine-to-alanine C-terminal substitution (HER2-V1255A) in HER2, abrogates the interaction with DLG1. In contrast, either PDZ domain 1 or 3 within Dlg1 is required for interaction with HER2. Moreover, HER2-Δ1250-1255- or HER2-V1255A-expressing MCF10A human breast epithelial cells show the reduction of mesenchymal-to-epithelial transition (EMT) proteins including vimentin and Snail. In addition, HER2-wt cells promotes EMT as shown by down-regulation of E-cadherin and upregulation of vimentin and Snail. Taken together, these results indicate that C-terminal PDZ binding domain in HER2 modulates biological functions of HER2. These results reveal new insight into the structure and function of HER2, which may guide the design and development of new anti-cancer targets to treat HER2-positive breast cancer. Citation Format: Dong Lin, Xiaodi Zhang, Angela Eldridge, Ming Fan, Cheikh Menaa, Jianjian Li. Her2 C terminal PDZ binding domain interacting with DLG1 is required for radioresistance through induction of EMT and loss of epithelial polarity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4408. doi:10.1158/1538-7445.AM2014-4408

Immune tolerance towards malignant cells is responsible for tumor progression and metastases. Despite their immunogenicity, cancer cells developed sophisticated mechanisms by which they are able to neutralize and/or evade the immune-surveillance. Recently, the overexpression of CD47 has been suggested to participate in the immune escape mechanism in several malignant diseases. However, mechanisms involved in CD47 expression, especially in response to anticancer agents are not yet known. In this study, we found that CD47 is overexpressed in breast cancer cells (MCF7, MDA-231, 4T1 cells) compared to non-transformed breast epithelial cells (MCF10A) and this expression is further enhanced in response to radiation (5Gy). The expression of CD47 was found to be regulated by NF-κB through a specific responsive element in the CD47 promoter as assessed by gene deletion and ChiP assays. Furthermore, using syngeneic orthotopic 4T1 mouse model, we found that CD47 is critical for both tumor burden and metastases, since anti-CD47 (100 μg/day/mice) was able to reduce tumor growth when the antibody was injected during 4T1 inoculation (day 0) or 15th after tumor growth. Consequently, tumor size and lesions were reduced and mouse survival was increased in the presence of anti-CD47 and this effect was further enhanced with radiation (4 Gy/day for 5 days). Taken together, these data suggest that CD47 expression is a marker for tumor aggressiveness and resistance to anti-cancer agents and therefore could be a potential target to treat metastatic and recurrent tumors. Citation Format: Cheikh Menaa, Ming Fan, Hsien-Chen Lu, Aris Alexandrou, Shuaib Juma, Julian Perks, Jian Jian Li. The dynamic change of CD47 expression promotes tumor burden, metastases and resistance of breast cancer cells to radiotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4963. doi:10.1158/1538-7445.AM2013-4963

Although the effectiveness of anti cancer therapies has improved dramatically, breast cancer (BC) remains the second leading cause for cancer-related death among women in the western world [1]. The observed improvement in the survival rate and remission for primary tumors are essentially related to the advances in early diagnosis with more sensitive imaging technology [2,3]. However, the death rate remains unacceptably high for BC patients due to hard-to-treat metastatic and recurrent tumors, necessitating new, effective approaches and anticancer agents. Accumulating evidence suggests that cancer stem cells (CSCs), which are present in many cancers including BC, mediate tumor metastasis and contribute to relapse due to their resistance to current conventional therapies [4,5]. These unique cells possess stem cell-like characteristics, such as the capacity of self-renewal, which makes the tumor capable of regenerating its entire bulk. CSCs are resistant to proapoptotic factors, rendering them a formidable adversary to anticancer agents. In part, this is related to their quiescence capacity, which holds them in a standby mode in their niche microenvironment, sheltering them from radiation and other anticancer agents, since these agents are effective only on highly proliferative cells [6,7]. In addition to their powerful DNA repair machinery, breast CSCs (BCSCs) express ALDH, which is suspected to play a part in their death-resistance phenotype by being involved with cell detoxification machinery [8,9]. Thus, BCSCs represent a major challenge in the battle against BC, and an opportunity to develop more effective target to treat metastatic lesions.

Purpose:Tounderstand the role ofHER2-associated signalingnetwork inbreast cancer stemcells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2 /low breast cancers. Experimental Design: HER2-expressing BCSCs (HER2þ/CD44þ/CD24 ) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2þ/CD44þ/CD24 /low versus HER2 /CD44þ/CD24 /low BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program. Results: Compared with HER2-negative BCSCs, HER2þ/CD44þ/CD24 /low cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2þ/CD44þ/CD24 /low cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/ MS-MS of HER2þ/CD44þ/CD24 /low versus HER2 /CD44þ/CD24 /low BCSCs reported a unique HER2associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2–STAT3 network was identified. Conclusion: This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2 /low breast cancer. Clin Cancer Res; 1–14. 2012 AACR.

Abstract Introduction Central and peripheral clocks generate self-sustained circadian rhythms that are intimately involved in metabolic and physiological processes. Circadian deregulation is associated with many human diseases including cancer, diabetes, and neurological disorders. Epidemiological studies have shown that disruption in circadian rhythms, such as nocturnal workers, is linked to an increased cancer risk including breast carcinoma. This effect is likely related to the perturbation of hormonal and cytokine circulation caused by deregulated circadian genes affecting cell survival and proliferation. Mice with gene deletion of Per2, a core component of the circadian rhythm, show higher incidence of lymphoma. Moreover, the absence of Per2 altered the levels of c-Myc and Cyclin D, two genes involved in the homeostasis of cell proliferation. Recent work demonstrates that Cyr, a partner of Per2, regulates cell death in response to chemotherapy agents. However, the exact function of Per2 signaling pathway in maintaining cell homeostasis against environmental stress conditions, such as low dose ionizing radiation, has not been elucidated. Methods and Results Our current studies aim to elucidate the contribution of PER2 in the low-dose radiation-induced radioadaptive response of human mammary epithelial cells (MCF10A). MCF10A and breast cancer cells (MCF7) treated with LDIR at 4-hour intervals (i.e., 0, 4, 8, 12, 16, 20, 24) showed highest PER2 expression at 8 hours in contrast to the absence of PER2 in MCF7 cells. To examine whether LDIR-induced Per2 expression is responsible for the cell death adaptation, MCF10 cells transfected with either Per2 siRNA or scramble were exposed to different doses of IR (i.e., 10cGy, 10cGy + 5Gy, and 5Gy). We found that knockdown of Per2 gene expression caused ablation of the LDIR-induced radioadaptive response with a significant increase in apoptosis of irradiated samples compared to controls. This finding was translated by reduced clonogenic survival and increased apoptosis assessed by flow cytometry analysis. Furthermore, mechanistic analysis showed that phospho-glycogen synthase kinase-3ß (p-GSK3ß) activation mediated LDIR-induced PER2 expression, and subsequently the adaptive protection of human mammary epithelial cells. Therefore, an enhanced interaction between p-GSK3ß and PER2 plays an active role in LDIR-induced radiation protection. This data represents the first experimental evidence that Per2 plays a critical role in protecting human epithelial cells in environmental stress conditions. Conclusion Induction of Per2 by LDIR treatment protects human breast epithelial cells against subsequential exposure to high dose radiation-induced genotoxic effects. Per2-mediated radioprotection requires p-GSK3ß activation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2168. doi:1538-7445.AM2012-2168

Abstract Despite a recent trend toward improvement, breast cancer (BC) mortality remains high due to resistance of recurrent tumors. A major cause of recurrence is likely a micro-metastatic seeding of cells that developed resistance by activating intrinsic or acquired survival pathways. In this regard, HER2 expression and cancer stem cells (CSCs) are the major cause for recurrence and targeting HER2 is well considered in therapeutic regiments. However, clinical data showed that anti-HER2 also benifit HER2−/low BC patients, suggesting that tumor cells may acquire HER2 gene activation to survive. We have previously reported that NF-κB mediates radioresistance by controlling HER2 gene. Herein, we identified HER2-expressing BC stem cells (HER2+/CD44+/CD24−/low) in radiation-resistant HER2−/low BC cells, MCF7/C6. Matrigel invasion, tumor sphere formation and radioresistance were enhanced in the HER2 mediating radioresistant cells. About 10% of the CD44+/CD24−/low cells sorted from MCF7/C6 cells or irradiated xenografts showed co-expression of HER2/CD44, ALDH and high tumorigenicity compared to HER2−/CD44+/CD24−/low cells, suggesting that the induction of HER2 expression further increased the BCSC aggressiveness. In fact, clinical data indicates that cells co-expressing HER2/CD44 were increased in recurrent tumors (84.6%) compared to the primary tumors (57.1%). Proteomics analysis identified a unique profile of proteins including DNA repair, mitochondrial function, redox, mTOR pathways that may govern the resistance of HER2+/CD44+/CD24−/low CSCs. These results demonstrate that HER2-mediated BC resistance can develop at the level of BCSC. Thus, HER2-expressing BCSCs may serve as an effective target to treat BC recurrence even from primary HER2−/low tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5724. doi:1538-7445.AM2012-5724