HER2-positive Breast Cancer Tissues Research Articles

Establishment of a Novel Cancer-Specific Anti-HER2 Monoclonal Antibody H2Mab-250/H2CasMab-2 for Breast Cancers.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 monoclonal antibody, named H2Mab-250/H2CasMab-2 (IgG1, kappa). H2Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H2Mab-250 did not react with nontransformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs, such as H2Mab-119 and trastuzumab reacted with both cancer and normal epithelial cells. Immunohistochemical analysis demonstrated that H2Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared with H2Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H2Mab-250. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

High contrast breast cancer biomarker semi-quantification and immunohistochemistry imaging using upconverting nanoparticles.

Breast cancer is the second leading cause of cancer death in women. Current clinical treatment stratification practices open up an avenue for significant improvements, potentially through advancements in immunohistochemistry (IHC) assessments of biopsies. We report a high contrast upconverting nanoparticles (UCNP) labeling to distinguish different levels of human epidermal growth factor receptor 2 (HER2) in HER2 control pellet arrays (CPAs) and HER2-positive breast cancer tissue. A simple Fourier transform algorithm trained on CPAs was sufficient to provide a semi-quantitative HER2 assessment tool for breast cancer tissues. The UCNP labeling had a signal-to-background ratio of 40 compared to the negative control.

Abstract P5-02-53: P95HER2 Expression in HER2-Positive Breast Cancer

Abstract Background: -HER2-positive breast cancer subtype accounted for around 15-20% of all breast cancer. -The introduction of HER2-targeted therapy such as trastuzumab and pertuzumab has remarkably increased the patients’ prognosis of HER2-positive breast cancer. -However, resistance exists due to impaired drug binding to HER2 receptor and constitutive activation of HER2 downstream signaling pathways. -P95HER2 isoform is a truncated form of HER2 that retains the C terminal domain but lacks an N terminal trastuzumab binding site, leading to trastuzumab resistance in HER2-positive breast cancer. -A new P95HER2 antibody is developed to target the extracellular domain of p95HER2 in formalin-fixed paraffin-embedded (FFPE) HER2-positive breast cancer tissues by using hematoxylin and eosin (HE) staining method. Objectives: To evaluate the expression of P95HER2 and its clinicopathological characteristics in HER2-positive breast cancer. Methods: We assessed 68 HER2-positive patients (IHC 3+ or IHC 2+/in situ hybridization [ISH]+) from Fudan University Shanghai Cancer Center (FUSCC) who underwent breast cancer surgery and were treated with adjuvant chemotherapy (taxane or anthracycline or combination) plus trastuzumab from 2014 to 2016. P95HER2 HE antibody is provided by Simcere Pharma. In this study, we compared 27 patients with primary trastuzumab resistance with 41 non-relapse breast cancer patients. 14 patients have not received trastuzumab targeted therapy. P95HER2 staining of either 1+, 2+ or 3+ observed in any tumor area in HE slides was considered to be P95 HER2 positive. Chi-square test was used to determine the relationship between P95HER2 expression of patients’ characteristics. The main outcome measures were disease free-survival (DFS), distant disease-free survival (DDFS) and overall survival (OS) by using log-rank test. Univariable and multivariable Cox regression analyses were used to identify independent factors related to prognosis. Results: From 2014 to 2016, we assessed the expression of P95HER2 expression in 68 HER2 positive breast cancer patients from FUSCC. Median follow-up was 45 months. In our study, 19 (27.9%) were P95HER2 positive. P95HER2 positive expression rate is higher in premenopausal patients than in postmenopausal patients (68.4% vs 38.8%, P= 0.028). Univariable analysis showed that higher T-stage (P= 0.018), higher N-stage (P= 0.001) and P95HER2 positive expression (P= 0.033) were associated with worse DDFS. Multivariable analysis showed that higher T-stage (hazard ratio, 6.019; 95% CI, 1.205-30.078; P= 0.029) and P95HER2 positive (hazard ratio, 2.349; 95%CI, 1.03-5.358; P= 0.042) independently predicted worse DDFS. P95HER2 positive was significantly associated with shorter 5-year DDFS (42.1% vs 67.6%, P= 0.028), but has no significant difference in DFS (36.8% vs 59.5%, P= 0.072) and OS (74.8% vs 81.2%, P= 0.685). Conclusions: P95HER2 positive was found more in premenopausal patients and was associated with a higher metastasis rate, indicating that P95HER2 expression tends to be a more aggressive isoform type of HER2-positive breast cancer. P95HER2 may serve as a therapeutic target for anti-HER2 therapy. Citation Format: Chih Wan Goh, Liyi Zhang, Wei-Ru Chi, Bingqiu Xiu, Jiajian Chen, Wenqing Yang, Chunxia Ao, Jianxing Tang, Jingyan Xue, Yayun Chi, Jiong Wu. P95HER2 Expression in HER2-Positive Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-53.

Abstract P5-02-41: UBE2E3 promotes the progression of HER2-positive breast cancer and influences the efficacy of targeted therapy via EGFR stabilization

Abstract Background: In the past 20 years, the efficacy and prognosis of HER2-positive breast cancer have significantly improved. However, nearly 50% of patients still have residual invasive tumors after chemotherapy combined with dual-targeted neoadjuvant therapy, especially for those with disease progression during treatment. A lack of effective therapeutic regimens results from the failure of targeted therapy, whose heterogeneity is especially worthy of our attention. The aim of this study was to look for efficacy markers and investigate new drug-resistance mechanisms. Methods: Firstly, the high-throughput sequencing data from 81 patients who received neoadjuvant chemotherapy TCbH (paclitaxel + carboplatin + trastuzumab) was analyzed by the efficacy outcomes. They were divided into 8 patients with stable or progressive disease (SD/PD), 35 with partial response (PR), and 38 with pathological complete remission (pCR). Then, UBE2E3 was chosen from the different expression genes between SD/PD and pCR based on efficacy results and the weighted gene co-expression network (WGCNA). UBE2E3 clinical correlations were investigated using publicly available data from The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC), and UBE2E3 was validated using immunohistochemistry (IHC) on 200 HER2-positive breast cancer tissue chips. Further, the UBE2E3 knockdown and overexpression stable transfer cell lines were constructed, and the effects of UBE2E3 on cell proliferation, clone formation, and drug sensitivity were verified by live cell imaging, the CCK8 assay, plate cloning, and IC50 assays, respectively. The tumor growth of UBE2E3 in vivo was investigated by an in situ transplantation tumor assay in nude mice. Meanwhile, the p-RB assay of mouse tissues by IHC was used to explore the effect of UBE2E3 on cell proliferation. RNA-seq was used to screen the downstream molecules of UBE2E3. Western blotting was used to verify the results of bioinformatics analysis and to explore the downstream key molecules. The protease inhibitor MG132 and actinomycin CHX were used to look at the effect on the stability of the target protein. Immunoprecipitation and silver staining assays were used to find interacting proteins with the UBE2E3. Results: Ten hub-genes which were efficacy-related were identified by WGCNA analysis, in which UBE2E3 was highly expressed in the SD/PD group (p < 0.05). In HER2-positive breast cancer, high expression of UBE2E3 was associated with poor prognosis and decreased disease-free survival both in public data and Fudan University Shanghai Cancer Center (FUSCC) data [HR 2. 36, (1.25–4.47), p < 0.05]. The experimental results demonstrated that UBE2E3 promoted the proliferation of HER2-positive breast cancer cells, enhanced clone formation, and resisted lapatinib’s treatment in cellular phenotype; and that UBE2E3 promoted tumor growth in vivo and upregulated the expression of p-RB. The differentially expressed genes’ sets of the RNA-seq between overexpressed cell lines and control showed that overexpressing UBE2E3 activated the EGFR pathway. Further, an immunoblot assay confirmed that UBE2E3 positively regulated EGFR levels and activated the downstream MAPK pathway. The proteasome inhibitor MG132 and CHX assays showed that UBE2E3 could stabilize EGFR proteins. The co-immunoprecipitation and silver staining assays showed that UBE2E3 stabilized EGFR proteins by interacting with c-Cbl. Conclusion: UBE2E3 could negatively affect the efficacy of HER2-positive breast cancer therapy and is significantly associated with poor prognosis. UBE2E3 may serve as a potential marker of efficacy and prognosis for HER2-positive breast cancer in the future. Therapeutic efficacy is affected by UBE2E3, which binds to c-Cbl and causes upregulation of EGFR expression in vivo, which in turn causes the MAPK pathway to be activated and tumor growth to be pushed up. Citation Format: Pei Li, Wei-Ru Chi, Bingqiu Xiu, Qi Zhang, Liyi Zhang, Ming Chen, Jingyan Xue, Xiaoyan Huang, Yayun Chi, Jiong Wu. UBE2E3 promotes the progression of HER2-positive breast cancer and influences the efficacy of targeted therapy via EGFR stabilization [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-41.

CircEPSTI1 Promotes the Proliferation of HER2-Positive Breast Cancer Cells via circEPSTI1/miR-145/ERBB3 Axis.

Breast cancer is the most common type of cancer worldwide. There are great challenges in the prevention and treatment of breast cancer. In this study, we explored the molecular and biological mechanisms of circular RNA circEPSTI1 (has_circ_0000479) in the regulation of HER2-positive breast cancer cells. The expression of CircEPSTI1, microRNA miR-145, and ERBB3 in HER2-positive breast cancer cells was evaluated by qRT-PCR and western blot assays. Cell proliferation was assessed by CCK-8. Wound-healing and transwell migration assays were performed to evaluate cell migration. A transwell invasion assay was performed to detect cell invasion. The interaction of miR-145, circEPSTI1, and ERBB3 was confirmed bydual-luciferase reporter and RIP assays. CircEPSTI1 was upregulated in the HER2-positive breast cancer tissues and cells. Knockdown of circEPSTI1 inhibited SKBR3 and BT474 cell proliferation, migration, and invasion. Mechanistically, circEPSTI1 directly targeted miR-145, and miR-145 was a downstream mediator of circEPSTI1 in modulating the proliferation, migration, and invasion of SKBR3 and BT474 cells. ERBB3 was identified as a direct and functional target of miR-145 in HER2-positive breast cancer cells. Our findings demonstrate that circEPSTI1, an overexpressed circRNA in HER2-positive breast cancer, promotes the proliferation, migration, and invasion of SKBR3 and BT474 cells through the miR-145/ERBB3 axis.

Abstract 5127: Significance of Trop-2 in HER2-positive breast cancer

Abstract Purpose: The glycoprotein Trop-2 is frequently overexpressed in human malignancies and linked to worse outcome. A Trop2-targeting antibody-drug conjugate, sacituzumab govitecan, has been approved by the FDA for patients with unresectable locally advanced or metastatic triple-negative breast cancer. Trop-2 enhances Akt phosphorylation in numerous cancer cell lines, while the opposite findings are also reported suggesting the cell context-dependent effect of Trop-2. Blockade of PI3K/Akt pathway mediates anti-tumor mechanism of anti-HER2 agents. In this study, we aim to investigate the role of Trop-2 in HER2-positive breast cancer. Experimental design: The archival samples comprising different breast cancer subtypes were analyzed using immunohistochemical staining. The public databases, including, Metabric, KM plotter, ROC plotter, were used to verify the prognostic role and clinical significance of Trop-2 in HER2-positive breast cancer. In vitro tumor models, the transwell migration, invasion and sphere assays, were applied for evaluation of cancer progression. The apoptotic cells were determined by flow cytometry using Annexin V/propidium iodide double staining. Results: We analyzed a cohort of 986 breast cancer patients and found that HER2-positive breast cancer harbored higher Trop-2 protein expression than other breast cancer subtypes. Higher levels of Trop-2 and phospho-Akt in HER2-positive breast cancer tissues compared with normal tissues. Trop-2 expression was correlated with tumor stage and phospho-Akt level. HER2-positive breast cancer patients with high level of Trop-2 protein were linked to worse recurrence-free survival. Consistently, data from the public databases exhibited patients with higher level of Trop-2 transcript had shorter recurrence-free survival and overall survival of HER2-positive breast cancer. Overexpression of Trop-2 increased cell migration, invasion and sphere number in SK-BR-3 and BT-474 cells. In contrast, knockdown of Trop-2 suppressed cell motility and sphere formation. Trop-2-expressing cells had better cell viability compared to control cells in the presence of lapatinib or neratinib treatment. Lapatinib- and neratinib-induced cell apoptosis was rescued by Trop-2 overexpression. Notably, patients responded to anti-HER2 therapy had lower Trop-2 transcript expression than non-responders. Conclusion: These results suggested that the oncogenic role of Trop-2 in HER2-positive breast cancer and Trop-2 expression might confer resistance to anti-HER2 therapy. Citation Format: Chun-Yu Liu, Ji-Lin Chen, Pei-Yi Chu, Chun-Teng Huang, Wan-Lun Wang, Chi-Cheng Huang, Po-Han Lin, Ling-Ming Tseng. Significance of Trop-2 in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5127.

Abstract P5-10-02: A novel mechanism of phosphatase activation for EGFR by Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer cells

Abstract Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is correlated with poor prognosis. HER2-targeting drugs have been successful to treat HER2-positive breast cancer. However, the acquisition of the drug resistance with long-term use is still recognized. Here we report the novel molecular targets to treat HER2-positive breast cancer. HER2 is a key driving forces to proliferate HER2-positive breast cancer cells. Unlike other HER family members (EGFR, HER3, HER4), it recognizes no ligands and forms heterodimers with each other member, leading to the transduction of various cellular signals. Especially, the activated EGFR transactivates HER2, and the phosphorylation of both EGFR and HER2 strongly drives cell proliferation. In this study, we found that the degradation of an endosomal small GTPase, RhoB, by the ubiquitin ligase complex cullin-3 (CUL3)/KCTD10 is essential for both EGFR and HER2 phosphorylation in HER2-positive breast cancer cells. Depletion of CUL3 or KCTD10 drastically reduced the phosphorylation of EGFR and HER2, as well as HER2-positive breast cancer cell proliferation. As mechanisms, we identified a RhoB-interacting scaffold protein and an EGFR phosphatase from human protein arrays prepared by the wheat cell-free protein synthesis system, a unique technology of Ehime University. In HER2-positive breast cancer cells, we showed that the scaffold protein interacts with the EGFR phosphatase at the plasma membrane, which leads to inactivation of the EGFR phosphatase and inhibits EGFR dephosphorylation. Upon CUL3- or KCTD10-knockdwon, accumulated RhoB interacts with the scaffold protein, resulting in the release of active EGFR phosphatase. Using the METABRIC database, we next found that low expression of RhoB mRNA and high expression of scaffold protein correlate with poor prognosis for HER2-positive breast cancer patients. Additionally, high expression of scaffold protein was associated with poor prognosis in immunohistochemistry of HER2-positive breast cancer tissues. These data suggest that high expression of scaffold protein enhances cell proliferative signaling in HER2-positive breast cancers. The inhibitors of the scaffold protein/EGFR phosphatase interaction would be new therapeutic agents for HER2-positive breast cancer cells through EGFR dephosphorylation mediated by activation of the EGFR phosphatase. Citation Format: Kanako Nishiyama, Masashi Maekawa, Akari Murakami, Kaho Utsunomiya, Kana Takemoto, Erina Kusakabe, Haruna Noda, Reina Aoki, Kana Taguchi, Michiko Yamashita, Tomoya Nakagita, Jun Nakayama, Mami Chosei, Takeshi Kiyoi, Yoshiaki Kamei, Hiroyuki Takeda, Yasutsugu Takada, Shigeki Higashiyama. A novel mechanism of phosphatase activation for EGFR by Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-02.

Abstract PS19-27: Upregulation of SLC26A9 resulted in the development and progression of HER2 positive breast cancer via activating TGFβ signaling pathway

Abstract Background: Slc26a9 (Solute carrier family 26 member 9) is a member of Slc26a family of multifunctional anion transporters, which functions as Cl- channel (Liu et al., Front Physiol 2018). Our previous study showed that SLC26A9 deficiency results in the development and progression of gastric cancer, which demonstrated that the key role of SLC26A9 in the tumorigenesis first time (Liu et al., Gastroenterology 2018). However, what’s the role of SLC26A9 in the breast cancer (BC) onset is not clear. We therefore wondered whether Slc26a9 gene is involved in promoting breast carcinogenesis and its mechanisms. Methods: The tissue microarrays and IHC assay were used to detect the expression and clinic relevance of SLC26A9 in the human BC. Different BC cell lines were used to investigate the expression and function of SLC26A9 in the BC. SLC26A9 gene transfect and knockout experiment were performed to detect the regulator role of SLC26A9 in BC cell behaviors. Results: Compared with adjacent normal tissues, SLC26A9 expression was significantly increased in the BC tissue, SLC26A9 expression level in BC correlated with the differentiated state of BC and patient’s clinical outcome, indicating that SLC26A9 may be involved in BC pathogenesis and progression in humans and it might be a novel poor prognosis marker for BC. Moreover, HER2 positive BC tissues exhibited high SLC26A9 expression than HER2 negative BC tissues. Compared with normal mammary cell line, SLC26A9 was significantly upregulated in the all BC cell lines, with highest expression in HER2 enriched cell line SKBR3. Deletion of SLC26A9 in SKBR3 resulted in inhibition of BC cell proliferation, migration and invasion, but promotion of cell apoptosis, which were investigated by different cell function assays, including cell counting Kit-8, cell proliferation curve, Annexin VFITC/PI double staining, Wound-healing and transwell assays respectively. Moreover, SLC26A9 deficiency in SKBR3 caused alteration of epithelial mesenchymal transition (EMT) markers, including downregulation of N-cadherin, Snail1, Fibronectin and Vimentin, but increasement of E-cadherin and ZO-1, accompany with disrupting TGFβ signaling pathways, including downregulation of TGF-β1, p-Smad2 and p-Smad3. However, overexpression of SLC26A9 in SKBR3 resulted in inhibition of cell proliferation, migration and invasion, but promotion of cell apoptosis. Accompanying with activation of TGFβ pathway mediate EMT. Conclusions: Upregulation of SLC26A9 resulted in the development and progression of HER2 positive BC via activation of TGFβ signaling pathways. SLC26A9 might be a novel prognosis marker and therapeutic target for BC. Citation Format: Zhiyuan Ma, Xuemei Liu, Chengli Lu, Jiaxing Zhu, Xiaoming Cheng, Biguang Tuo, Taolang Li. Upregulation of SLC26A9 resulted in the development and progression of HER2 positive breast cancer via activating TGFβ signaling pathway [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-27.

24-Dehydrocholesterol reductase promotes the growth of breast cancer stem-like cells through the Hedgehog pathway.

Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24‐Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem‐like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway‐regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24‐induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem‐like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem‐like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer.

AK4 Promotes the Progression of HER2-Positive Breast Cancer by Facilitating Cell Proliferation and Invasion.

Breast cancer (BC) is a type of malignant tumor originating from the epithelial tissue of the mammary gland, and about 20% of breast cancers are human epidermal growth factor receptor 2 positive (HER2+), which is a subtype with more aggression. Recently, HER2-positive breast cancer is often accompanied by poor prognosis of patients, and targeted therapy showed a promising prospect. To combat this disease, novel therapeutic targets are still needed. Adenylate kinase 4 (AK4) is a member of the adenylate kinase family and is expressed in the mitochondrial matrix. AK4 is involved in multiple cellular functions such as energy metabolism homeostasis. Interestingly, AK4 was observed highly expressed in several tumor tissues, and the involvement of AK4 in cancer development was generally revealed. However, the possible role of AK4 on the growth and development of breast cancer is still unclear. Here, we investigated the possible functions of AK4 on the progression of HER2-positive breast cancer. We found the high expression of AK4 in HER2-positive breast cancer tissues from patients who received surgical treatment. Additionally, AK4 expression levels were obviously correlated with clinical-pathological features, including pTNM stage (P = 0.017) and lymph node metastasis (P = 0.046). We mechanically confirmed that AK4 depletion showed the obvious impairment of cell proliferation and invasion in MCF7 and MDA-MB-231 cells. AK4 also facilitates tumor growth and metastasis of HER2-positive breast cancer in vivo. In conclusion, we identified and mechanically confirmed that AK4 is a novel therapeutic target of HER2-positive breast cancer.

Epitope Mapping of Human HER2 Specific Mouse Monoclonal Antibodies Using Recombinant Extracellular Subdomains

Background:Human epidermal growth factor receptor 2 (HER2) is overexpressed in several human malignancies and numerous studies have indicated that it plays important roles in the development and maintenance of the malignant phenotype. Targeting of HER2 molecules with monoclonal antibodies (mAbs) is a promising therapeutic approach. However, anti-HER2 mAbs affect cancer cells differently, depending on the distinct epitopes which are the targets.Methods:Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues.Results:Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one reacted with subdomain I+II, 2 recognized either subdomain III or IV and 2 recognized subdomain III+IV. However, none of our mAbs recognized the subdomain II alone. The mAbs displayed either inhibitory or stimulatory effects on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implied better reactivity of the mAbs recognizing linear epitopes.Conclusions:Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, there seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.

STAT3-survivin signaling mediates a poor response to radiotherapy in HER2-positive breast cancers

Although radiotherapy resistance is associated with locoregional recurrence and distant metastasis in breast cancers, clinically relevant molecular markers and critical signaling pathways of radioresistant breast cancer are yet to be defined. Herein, we show that HER2-STAT3-survivin regulation is associated with radiotherapy resistance in HER2-positive breast cancers. Depletion of HER2 by siRNA sensitized HER2-positive breast cancer cells to irradiation by decreasing STAT3 activity and survivin, a STAT3 target gene, expression in HER2-positive breast cancer cells. Furthermore, inhibition of STAT3 activation or depletion of survivin also sensitized HER2-positive breast cancer cells to irradiation, suggesting that the HER2-STAT3-survivin axis is a key pathway in radiotherapy resistance of HER2-positive breast cancer cells. In addition, our clinical analysis demonstrated the association between HER2-positive breast cancers and radiotherapy resistance. Notably, we found that increased expression of phosphorylated STAT3, STAT3, and survivin correlated with a poor response to radiotherapy in HER2-positive breast cancer tissues. These findings suggest that the HER2-STAT3-survivin axis might serve as a predictive marker and therapeutic target to overcome radiotherapy resistance in HER2-positive breast cancers.

High mobility group B1 and N1 (HMGB1 and HMGN1) are associated with tumor-infiltrating lymphocytes in HER2-positive breast cancers.

Although the prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in HER2-positive breast cancers has been established, the drivers of TIL influx remain unclear. We tested whether potential triggers for this response could include high mobility group B1 and N1 (HMGB1 and HMGN1) proteins, which are immunogenic damage-associated molecular pattern molecules. We evaluated TILs and the immunohistochemical expression of HMGB1 and HMGN1 in 447 HER2-positive breast cancer tissues. Normal luminal cells exhibited nuclear expression of HMGB1 and HMBN1. The nuclear and cytoplasmic expression levels of HMG proteins showed a significant inverse correlation (rho=-0.150, p=0.001 for HMGB1; rho=-0.247, p<0.001 for HMGN1). Low levels of HMGB1 and HMGN1 nuclear expression were identified in 185 (41.4%) and 208 (46.5%) cases, respectively. High levels of cytoplasmic HMGB1 and HMGN1 expression were identified in 107 (23.9%) and 49 (11.0%) cases, respectively. High cytoplasmic expression of HMG proteins was significantly associated with a high histological grade, high levels of TILs, peritumoral lymphocytic infiltration, and tertiary lymphoid structures in HER2-positive breast cancer tissues. Tumors with low levels of cytoplasmic HMGB1 and HMGN1 showed significantly lower levels of TILs than those with high levels of each or both HMG proteins. However, the nuclear or cytoplasmic expression of either HMG protein was not found to be significantly associated with survival. High levels of cytoplasmic HMGB1 and HMGN1 protein expression correlated with high levels of TILs in HER2-positive breast cancers. The manipulation of HMGB1 and HMGN1 could represent an immunotherapeutic approach to promote TIL influx into a tumor.

Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast Cancer

Objective: Resistance to trastuzumab is a problem that remains to be solved in HER2-positive breast cancer. We aimed to characterize profiles of genetic and epigenetic alterations in cancer-related pathways in HER2-positive breast cancers, using biopsy tissue samples obtained from patients enrolled in a prospective neoadjuvant clinical trial. Methods: HER2-positive breast cancer tissue samples were collected and processed with the PAXgene Tissue System. A total of 24 breast cancers were analyzed. Genetic alterations of 409 cancer-related genes were analyzed by a bench-top next-generation sequencer. DNA methylation statuses were analyzed by a bead array with 485,512 probes. Results: The WNT pathway was potentially activated by aberrant methylation of its negative regulators, such as DKK3 and SFRP1, in 9 breast cancers. The AKT/mTOR pathway was activated by mutations of PIK3CA in 5 breast cancers. The Notch pathway was potentially activated by mutations of NOTCH1 and NOTCH2 in 4 breast cancers. The p53 pathway was inactivated by mutations of TP53 in 13 breast cancers and potentially by aberrant methylation of its downstream genes in 10 breast cancers. Cell adhesion was affected by mutations of CDH1 in 1 breast cancer. Conclusion: Genes involved in cancer-related pathways were frequently affected not only by genetic but also by epigenetic alterations in HER2-positive breast cancer.

MTDH mediates trastuzumab resistance in HER2 positive breast cancer by decreasing PTEN expression through an NFκB-dependent pathway.

BackgroundTrastuzumab resistance is almost inevitable in the management of human epidermal growth factor receptor (HER) 2 positive breast cancer, in which phosphatase and tensin homolog deleted from chromosome 10 (PTEN) loss is implicated. Since metadherin (MTDH) promotes malignant phenotype of breast cancer, we sought to define whether MTDH promotes trastuzumab resistance by decreasing PTEN expression through an NFκB-dependent pathway.MethodsThe correlations between MTDH and PTEN expressions were analyzed both in HER2 positive breast cancer tissues and trastuzumab resistant SK-BR-3 (SK-BR-3/R) cells. Gene manipulations of MTDH and PTEN levels by knockdown or overexpression were utilized to elucidate molecular mechanisms of MTDH and PTEN implication in trastuzumab resistance. For in vivo studies, SK-BR-3 and SK-BR-3/R cells and modified derivatives were inoculated into nude mice alone or under trastuzumab exposure. Tumor volumes, histological examinations as well as Ki67 and PTEN expressions were revealed.ResultsElevated MTDH expression indicated poor clinical benefit, shortened progression free survival time, and was negatively correlated with PTEN level both in HER2 positive breast cancer patients and SK-BR-3/R cells. MTDH knockdown restored PTEN expression and trastuzumab sensitivity in SK-BR-3/R cells, while MTDH overexpression prevented SK-BR-3 cell death under trastuzumab exposure, probably through IκBα inhibition and nuclear translocation of p65 which subsequently decreased PTEN expression. Synergized effect of PTEN regulation were observed upon MTDH and p65 co-transfection. Forced PTEN expression in SK-BR-3/R cells restored trastuzumab sensitivity. Furthermore, decreased tumor volume and Ki67 level as well as increased PTEN expression were observed after MTDH knockdown in subcutaneous breast cancer xenografts from SK-BR-3/R cells, while the opposite effect were found in grafts from MTDH overexpressing SK-BR-3 cells.ConclusionsMTDH overexpression confers trastuzumab resistance in HER2 positive breast cancer. MTDH mediates trastuzumab resistance, at least in part, by PTEN inhibition through an NFκB-dependent pathway, which may be utilized as a promising therapeutic target for HER2 positive breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-869) contains supplementary material, which is available to authorized users.

HER2Δ16 expression in HER2-positive breast cancer.

31 Background: HER2Δ16 isoform was detected in breast cancer and showed similar increased transforming activity to somatic mutation or deletion activated rat HER2 oncogene with 89% of HER2-positive breast cancer with this mutation developing local lymph node positive disease progression. This is the first study characterizing and determining the prevalence of HER2Δ16 isoform in patients from a NAPBC-accredited cancer center. Methods: 95 HER2-positive breast cancer tissue samples from 2002 to 2008 were obtained from Akron General Medical Center. Total RNA from cancer cells were isolated and RT-PCR was performed using primers to amplify the wild-type HER2 and HER2Δ16 isoform. DNA products from RT-PCR were then loaded in agarose gel electrophoresis with the HER2Δ16 isoform band being shorter compared to the wild type HER2. Results: Preliminary results from our ongoing study showed 39% (12 of 31) of our HER2 samples has HER2Δ16 isoform. Out of the 12 HER2Δ16 isoform samples, 17% are Stage I, 50% are Stage II, 25% are Stage III, and 8% are Stage IV breast cancer at diagnosis. Patient age ranges from 34 to 87, with 50% of the HER2Δ16 isoform patients having lymph–node positive disease at the time of diagnosis. This is in contrast to patients with HER2 wild type for which 26% (5 of 19) are diagnosed with Stage I disease and 42% have lymph node positive disease at the time of diagnosis. Conclusions: Transgenic mice that overexpressed rat HER2 oncogene with small extracellular domain deletion promoted transforming activity of this protein. HER2Δ16 isoform is detected in 39% of HER2 positive breast cancer based on our preliminary results with 50% having lymph node–positive breast cancer on initial diagnosis. Immunohistochemistry and FISH could not distinguish the wild type HER2 from the HER2Δ16 isoform. Further study is recommended for possible prognosticating utility of HER2Δ16 isoform in breast cancer.

The prognostic value of Her4 receptor isoform expression in triple-negative and Her2 positive breast cancer patients

BackgroundNot only four but rather seven different human epidermal growth factor receptor related (Her) receptor tyrosine kinases (RTKs) have been described to be expressed in a variety of normal and neoplastic tissues: Her1, Her2, Her3, and additionally four Her4 isoforms have been identified. A differential expression of Her4 isoforms does not, however, play any role in either the molecular diagnostics or treatment decision for breast cancer patients. The prognostic and predictive impact of Her4 expression in breast cancer is basically unclear.MethodsWe quantified the Her4 variants JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, and JM-b/CYT2 by isoform-specific polymerase chain reaction (qPCR) in (i) triple-negative, (ii) Her2 positive breast cancer tissues and (iii) in benign breast tissues.ResultsIn all three tissue collectives we never found the JM-b/CYT1 or the JM-b/CYT2 isoform expressed. In contrast, the two JM-a/CYT1 and JM-a/CYT2 isoforms were always simultaneously expressed but at different ratios. We identified a positive prognostic impact on overall survival (OS) in triple-negative and event-free survival (EFS) in Her2 positive patients. This finding is independent of the absolute JM-a/CYT1 to JM-a/CYT2 expression ratio. In Her2 positive patients, Her4 expression only has a favorable effect in estrogen-receptor (ER)-positive but not in ER-negative individuals.ConclusionIn summary, JM-a/CYT1 and JM-a/CYT2 but not JM-b isoforms of the Her4 receptor are simultaneously expressed in both triple-negative and Her2 positive breast cancer tissues. Although different expression ratios of the two JM-a isoforms did not reveal any additional information, Her4 expression basically indicates a prolonged EFS and OFS. An extended expression analysis that takes all Her receptor homologs, including the Her4 isoforms, into account might render more precisely the molecular diagnostics required for the development of optimized targeted therapies.

P1-12-23: HER4 Coexpression Is Associated with Improved Recurrence Free Survival in HER2−Positive, Herceptin Treated Patients.

Abstract Background: Individual therapy efficiency of HER2−positive metastatic and pre-metastatic breast cancer patients varies significantly and spans from effectual responsiveness over acquired insensitivity to complete resistance from the outset. Thus no predictive information can be deduced from HER2 diagnostics so that molecular biomarkers indicative for sensitivity / resistance to Herceptin are needed to be identified. The HER2 related HER4-receptor has been shown to have ambivalent (pro-apoptotic or pro-proliferative) activity and consequently represents a prime candidate to affect HER2 activity under Herceptin treatment. We retrospectively analyzed potential her4 gene amplification and HER4 protein expression in HER2−positive, Herceptin treated patients. Patient's overall and recurrence free survival was evaluated as a function of HER2/HER4 expression. Methods: Using dual color Fluorescence in-situ Hybridization (FISH probes, Zytovision, Bremerhaven, Germany) and qPCR (LC480, Roche, Penzberg, Germany) we quantitatively investigated primary breast cancer tissues from nearly 50 (FISH) and 160 (PCR) patients who received Herceptin treatment. We quantified the her4 gene copy numbers and evaluated the protein expression profile of all four known HER4 isotypes (JM-a/CYT1, JM-a/CYT2, JM-b/CYT1, JM-b/CYT2). Results: FISH analysis revealed a positive and independent prognostic marker in Herceptin treated breast cancer patients with respect to overall survival. Moreover by quantitative PCR analysis we found a significant variability of HER4 protein expression (JM-a/CYT1 and JM-a/CYT2; no JM-b isotypes) in HER2 positive breast cancer tissues, whereas HER2/HER4 positive patients show a significant better recurrence free survival compared to HER2 positive but HER4 negative patients (p = 0,003). Conclusions: HER4 has been demonstrated to potentially exert tumor suppressing activity and in turn to have a favourable impact on the course of breast cancer disease. We show here that HER4 expression prolongs in particular recurrence free survival of Herceptin treated patients which indicates a functional integration of HER4 into anti-HER2 targeting. Complementing functional studies allowing for isotype specific function of HER4 will elucidate the special role of this receptor tyrosine kinase in the context of Herceptin treatment and might facilitate individualized anti-ErbB-receptor targeting with higher efficiency. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-23.

Calpain regulates sensitivity to trastuzumab and survival in HER2-positive breast cancer.

Abstract Abstract #2018 Background: Overexpression of HER2 is associated with an aggressive cancer and poor prognosis for patients. HER2-positive patients are treated with trastuzumab; however resistance is common. Mechanisms that regulate activity of HER2 or downstream kinase AKT are involved in regulation of survival. Calpains are cysteine proteases involved in transmission of signals during growth, motility and transformation in cancer. Mechanisms by which calpain regulates transmission of signals in HER2-positive breast cancer are not known. Here we investigate possibility that calpain regulates survival in HER2-positive breast cancer.&amp;#x2028; Material and Methods: Activity of calpain in parental or resistant skbr3 cells was measured alone or in presence of inhibitors under conditions of adhesion or stimulation with trastuzumab using a fluorescently labeled substrate. To investigate regulation of HER2, assays were performed by mixing purified proteins or cell lysates with calpain1. Activity of HER2 was analyzed using phosphotyrosine-specific antibodies in Western blots while cleavage using antibody specific to the C-terminus. HER2-positive cancer tissue or lysates of cell lines were analyzed for presence of C-terminal fragments. Cells were transfected with the plasmid expressing catalytically inactive mutant of calpain1, selected for resistance to G418 and used in experiments to analyze the activity of HER2 and AKT. Sensitivity to trastuzumab was determined by measuring survival in presence of trastuzumab in a 96 well plate assay.&amp;#x2028; Results: In skbr3 cells, activity of calpain increased following adhesion or stimulation with trastuzumab and was inhibited by inhibitors. Calpain induced cleavage of C-terminal domains and reduced activity in full length HER2. C-terminal fragments were detectable in the lysates of HER2-positive breast cancer tissue, cell lines and also overproduced by overexpression of calpain1. Conversely, in cells expressing catalytically inactive calpain1 the activity of HER2 and AKT was increased correlating with increased survival on fibronectin in the presence of trastuzumab. As another model for calpain-induced regulation of HER2 in mechanisms of resistance, we analyzed regulation in trastuzumab-resistant skbr3 cells. Compared to parental in resistant cells adhesion- or trastuzumab-dependent activity of HER2 was increased whereas cleavage was reduced suggesting mechanisms that regulate generation of C-terminal fragments during transmission of signals were altered. Consistenly, adhesion-dependent activity of calpain was reduced whereas trastuzumab failed to stimulate activation.&amp;#x2028; Conclusion: These data are consistent with possibilities 1) HER2 is a direct target of calpain, 2) calpain induces cleavage of the cytoplasmic region during transmission of signals to regulate activity and integrity of HER2, 3) calpain-dependent mechanisms are involved in coupling HER2 to survival and 4) inhibition of calpain during adhesion-dependent transmission of signal or in the presence of trastuzumab allows increased activity of HER2 contributing to resistance. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2018.

Selection and Characterization of Her2 Binding-designed Ankyrin Repeat Proteins

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.