Knockdown Of HER2 Research Articles

Effect of neratinib on HER2 mRNA stability via hsa-miR-23a-5p and efficacy of CDK4/6 inhibitors in HER2-low breast cancer.

e12550 Background: Traditional HER2-negative breast cancer sometimes includes HER2-low components, and CDK4/6 inhibitors are the first- and second-line treatments for advanced HER2-low breast cancer. Nonetheless, little is understood about how the stability of HER2 mRNA in HER2-low breast cancer affects the effectiveness of CDK4/6 inhibitor therapy and the associated intervention techniques. Methods: In order to investigate the impact of HER2-low on the effectiveness of CDK4/6 inhibitor in treating breast cancer, we first used HER2-0 and HER2-low breast cancer cells/mice as the study object and assessed the anti-proliferation effect of CDK4/6 inhibitor using CCK8 technique and monoclonal formation experiment. Second, we increased the treatment of anti-HER2 inhibitor neratinib and assessed its impact on the efficacy of HER2 low breast cancer CDK4/6 inhibitors. By reducing the expression of HER2 in HER2 low breast cancer cells, we analyzed and determined whether HER2 is the primary pathway that influences the poor efficacy of HER2 low breast cancer CDK4/6 inhibitors; At last, actinomycin D was used to detect the mRNA stability of HER2 in the treatment of HER2-low breast cancer by neratinib against CDK4/6 inhibitors, and then microRNA sequencing and luciferase reporter gene detection technology were used. To investigate the specific mechanism of action of neratinib on the mRNA stability of HER2 in the treatment of HER2-low breast cancer by CDK4/6 inhibitors. Results: Our study found that treatment with CDK4/6 inhibitors was significantly less effective against HER2-low breast cancer compared to HER2-0 subtype (P=0.005). Notably, in HER2-low subtype cells, HER2 knockdown significantly inhibited cell proliferation (P<0.001), G1 phase cell cycle arrest (P=0.001), and downregulated cyclin D1 expression and CDK4 protein levels (P=0.007, P=0.003). Second, it was found that in the cell and animal models of HER2-low breast cancer, the addition of the CDK4/6 inhibitor drug neratinib effectively reduced the stability of HER2 mRNA (P=0.001), inhibited the cell cycle of G1 phase (P=0.005). In addition, we observed that lowering the dose of neratinib to 1/2 (20 mg/kg) remained effective and well tolerated (P=0.002). Finally, we found that neratinib can reduce the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3'UTR region of HER2 (P<0.001), thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. Conclusions: We found that low expression of HER2 reduced the efficacy of CDK4/6 inhibitors in HER2-low breast cancer; Neratinib reduces the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3 'UTR region of HER2, thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. These findings provide a tolerable and effective two-drug combination regimen for patients with clinical HER2-low breast cancer.

Abstract PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

Abstract 22: Development and characterization of human tumor specific cytotoxic lymphocytes for preclinical drug development

Abstract Heterogeneity within a tumor allows it to better adapt to changing conditions and can have therefore a negative impact on a patient’s prognosis, response to treatment and overall health. Breast cancer is a highly heterogeneous disease caused by distinct genetic mutations in mammary epithelial cells, resulting in significantly different forms of the disease and a need for highly personalized treatment schemes for patients. Hence, it is important to develop and improve (immuno)therapies for breast cancer patients. Adoptive T-cell therapy with autologous CD8+ cytotoxic T cell lymphocytes (CTL) is a promising method that has already shown promising effects in melanoma patients. Unfortunately, there is almost no information about the use in breast cancer patients. We developed an in vitro protocol to generate tumor-specific CTLs by priming on HER2+ breast cancer cell lines JIMT-1 and SKBR-3. Subsequently, these primed CTLs were tested in 2D and 3D immune cell killing assays in a life cell imaging device in combination with an endpoint viability assay. The activity was compared with unspecific CD8+T cells activated by phytohemagglutinin (PHA) or αCD2/αCD3/αCD28, HER2-peptide specific T cells and HER2-CAR T cells serving as a positive control. We investigated the feasibility to freeze CTLs for use in subsequent studies and validated the relevance of HER2 as the main druggable target in the tumor lines using an siRNA approach. The CTLs were most effective on JIMT-1 in a 3D setting, whereas the SKBR-3 CTLs displayed similar activity in 2D and in 3D. The respective CTLs were tumor line specific, as SKBR-3 CTLs did not kill efficiently the JIMT-1 cells and vice versa. The same CTLs displayed the same efficacy pattern after thawing but less pronounced. Thus, within one experimental set-up it is recommended to use either one. The Her2 knockdown in the target cells reduced the killing activity of the CTLs slightly. However, this effect was not statistically significant proving the importance of CTLs as treatment option as well as neoantigens as targets in drug development. In summary, the CTLs proved to capture the respective tumor heterogeneity showing high killing rates in both SK-BR3 and JIMT-1 being more active than HER2-peptide specific T cells. The availability of CTLs in a preclinical setting facilitates screening approaches for combinational studies. Beyond that they can serve as starting material to develop cancer vaccines and identify highly immunogenic neoantigens. Citation Format: Ina Rohleff, Lea Gröne, Kanstantsin Lashuk, Julia B. Schueler. Development and characterization of human tumor specific cytotoxic lymphocytes for preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 22.

Role of human epidermal growth factor receptor 3 in treatment resistance of anaplastic lymphoma kinase translocated non-small cell lung cancer

ABSTRACT Background ALK tyrosine kinase inhibitors (TKI) have revolutionized the treatment of ALK+ non-small cell lung cancer (NSCLC), and therapy resistance occurs in virtually all patients. Multiple TKI resistance mechanisms have been characterized, including ERBB receptor coactivation. In this study, we investigated the role of HER3 in ALK TKI resistance. Methods In vitro studies were carried out using ALK+ NSCLC cell lines H3122, H2228, and DFCI032. Pharmacological co-targeting of ALK and HER3 was investigated with ALK and ERBB TKIs, and HER3 knockdown was achieved using the CRISPR-Cas9 system. Co-localization of ALK and HER3 was investigated by immunoprecipitation (IP) and proximity ligation assay (PLA) in vitro and in vivo using six ALK+ NSCLC tumor samples. Results In all tested cell lines, combined targeting with ALK and pan-ERBB TKI resulted in marked inhibition of colony formation and long-term (72 h) downregulation of pAKT levels. HER3 knockdown resulted in multiple effects on ALK+ cell lines, including the downregulation of ALK expression and visible morphological changes (H2228). Co-immunoprecipitation (IP) and proximation ligation assay (PLA) experiments provided evidence that both ALK and HER3 could interact in vitro, and this finding was verified by PLA using ALK+ NSCLC tumors. Conclusions This study provides evidence that HER3 may mediate TKI resistance in ALK+ NSCLC. Interestingly, we were able to show that both translocated ALK and HER3 could interact. Joint targeting of ALK and HER3 could be further investigate in ALK+ NSCLC.

HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel

BackgroundTriple negative breast cancer (TNBC) represents a significant clinical challenge. Chemotherapy remains the mainstay for a large part of TNBC patients, whereas drug resistance and tumor recurrence frequently occur. It is in urgent need to identify novel molecular targets for TNBC and develop effective therapy against the aggressive disease.MethodsImmunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. IncuCyte system was utilized to monitor cell growth and migration. LIVE/DEAD Cell Imaging was to detect live and dead cells. HER3 recognition by our anti-HER3 monoclonal antibody (mAb) 4A7 was verified by ELISA, flow cytometry, and co-immunoprecipitation assays. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our mAb 4A7 could potentiate the antitumor activity of paclitaxel in vivo.ResultsElevated expression of HER3 was observed in approximately half of the TNBC specimens and cell lines tested. Analyses of TCGA databases found that the TNBC patients with high HER3 mRNA expression in the tumors showed significantly worse overall survival (OS) and relapse-free survival (RFS) than those with low HER3 expression. Specific knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Our mAb 4A7 abrogated heregulin (a ligand for HER3), but not SDF-1 (a ligand for CXCR4)-induced enhancement of TNBC cell migration. Combinations of 4A7 and the EGFR-tyrosine kinase inhibitor (TKI) gefitinib dramatically decreased the levels of phosphorylated HER3, EGFR, Akt, and ERK1/2 in TNBC cells and potently induced growth inhibition and cell death. Moreover, 4A7 in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo.ConclusionsOur data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of gefitinib or paclitaxel in TNBC.

Abstract A007: Modulation of HER2 as a mechanism for AR expression

Abstract Background: Intense neoadjuvant androgen deprivation therapy (inADT) is an emerging treatment paradigm for localized high-risk prostate cancer, but in certain patients a subset of tumors will recur after surgery. In tumors with endogenously low androgen receptor (AR) activity, expression of the EGFR family protein HER2 was enriched. We sought to investigate the role of HER2 in driving the proliferation of prostate cancer under androgen-depleted environments. We hypothesized that overexpression of HER2 would decrease AR activity, while its knockdown would conversely increase AR expression beyond baseline levels. Methods: The AR-expressing 22Rv1 and LNCaP prostate cancer cell lines were cultured and plated for transfection. HER2 overexpression was achieved using an exogenous HER2 (ERBB2) plasmid, while HER2 knockdown was performed using siRNA, alongside controls for both groups. Cell lysates were collected at 24-, 48-, and 72-hour time points. Western blotting was performed to examine HER2, AR, PSA, and actin expression. Results: Western blot analysis confirmed successful overexpression and knockdown of HER2, using an exogenous HER2 plasmid and siRNA, respectively. In 22Rv1 and LNCaP cells overexpressing HER2, AR (and AR-V7 in 22Rv1 cells) levels were decreased compared to the control cells. Conversely, 22Rv1 and LNCaP cells with knockdown of HER2 exhibited elevated AR expression. Biological replicates to confirm these findings are in progress. Additional studies will also examine the impact of HER2 signaling and activity on downstream pro-proliferative pathways. Conclusions: Manipulation of HER2 expression highlights a potential role of EGFR family proteins as a mechanism for prostate cancer growth in androgen-deprived environments. Future studies will validate this finding at the RNA level using qPCR and probe downstream targets of HER2 using western blotting. These preliminary findings have implications for the treatment of advanced tumors for which inADT is not a viable option. Targeting HER2 and its downstream signaling pathways represent an intriguing mechanism to overcome resistance to androgen deprivation therapy in newly-diagnosed, treatment-naïve patients. Citation Format: Daniel K.Y. Low, Scott Wilkinson, Isaiah M. King, Shana Y. Trostel, Adam G. Sowalsky. Modulation of HER2 as a mechanism for AR expression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A007.

Effect of HER2-targeted therapy on PDX and PDX-derived organoids generated from HER2-positive salivary duct carcinoma.

We previously established a patient-derived xenograft (PDX) model, patient-derived organoids (PDOs), and PDX-derived organoids (PDXOs) for salivary duct carcinoma (SDC). Using these models, this study examined the therapeutic effect of human epidermal growth factor receptor 2 (HER2) blockade on HER2-positive SDC. The therapeutic effect of lapatinib was assessed in SDC PDXOs with regards to cell growth, receptor/downstream signaling molecule expression, phosphorylation levels, and apoptosis. Effect of lapatinib treatment was evaluated in vivo in SDC PDX mice. The siRNA knockdown of HER2 and lapatinib suppressed cell proliferation in SDC PDXOs. Lapatinib inhibited the phosphorylation of HER2 and its downstream targets, and induced apoptosis in SDC PDXOs. Lapatinib also significantly reduced tumor volumes compared with that of the control in SDC PDX mice. For the first time, we demonstrated the efficacy of anti-HER2 therapy in HER2-positive SDC using preclinical models of SDC PDX and PDXO.

Characterization of Expression and Function of the Formins FHOD1, INF2, and DAAM1 in HER2-Positive Breast Cancer.

Human epidermal growth factor receptor 2 (HER2)-targeted therapies, such as trastuzumab, benefit patients with HER2-positive metastatic breast cancer; however, owing to traditional pathway activation or alternative signaling, resistance persists. Given the crucial role of the formin family in shaping the actin cytoskeleton during cancer progression, these proteins may function downstream of the HER2 signaling pathway. Our aim was to uncover the potential correlations between formins and HER2 expression using a combination of public databases, immunohistochemistry, and functional in vitro assays. Using online databases, we identified a negative prognostic correlation between specific formins mRNA expression in HER2-positive cancers. To validate these findings at the protein level, immunohistochemistry was performed on HER2 subtype breast cancer tumors to establish the links between staining patterns and clinical characteristics. We then knocked down individual or combined formins in MDA-MB-453 and SK-BR-3 cells and investigated their effects on wound healing, transwell migration, and proliferation. Furthermore, we investigated the effects of erb-b2 receptor tyrosine kinase 2 (ERBB2)/HER2 small interfering RNA (siRNA)-mediated knockdown on the PI3K/Akt and MEK/ERK1 pathways as well as on selected formins. Our results revealed that correlations between INF2, FHOD1, and DAAM1 mRNA expression and ERBB2 in HER2-subtype breast cancer were associated with worse outcomes. Using immunohistochemistry, we found that high FHOD1 protein expression was linked to higher histological grades and was negatively correlated with estrogen and progesterone receptor positivity. Upon formins knockdown, we observed effects on wound healing and transwell migration, with a minimal impact on proliferation, which was evident through single and combined knockdowns in both cell lines. Notably, siRNA-mediated knockdown of HER2 affected FHOD1 and INF2 expression, along with the phosphorylated Akt/MAPK states. Our study highlights the roles of FHOD1 and INF2 as downstream effectors of the HER2/Akt and HER2/MAPK pathways, suggesting that they are potential therapeutic targets in HER2-positive breast cancer.

Pan-HER inhibitors overcome lorlatinib resistance caused by NRG1/HER3 activation in ALK-rearranged lung cancer.

Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) with a broad coverage against ALK mutations, has demonstrated dramatic effects in patients with ALK-rearranged lung cancer. The mechanisms of acquired resistance to lorlatinib by secondary ALK compound mutations have recently been reported; however, resistance mechanisms other than secondary mutations remain unclear. Here, we investigated the molecular mechanisms of the acquired resistance in ALK-rearranged lung cancer cells in vitro. We established two different lorlatinib-resistant ALK-rearranged lung cancer cell lines (H3122LR and A925LLR) via long-term administration of lorlatinib. These resistant cells did not harbor the secondary ALK mutations and showed cross-resistance to the other kinds of ALK-TKIs (crizotinib or alectinib) compared with the parental cells; however, these resistant cells overexpressed the phosphorylated human epidermal growth factor receptor 3 (HER3) protein and the ligand of HER3 (neuregulin 1; NRG1). Pharmacological inhibition of HER3 with pan-HER inhibitors or genetic knockdown of HER3 with siRNA resensitized H3122LR and A925LLR cells to lorlatinib in vitro, indicating that H3122LR and A925LLR acquired resistance by NRG1/HER3 activation. These findings demonstrated that targeting NRG1/HER3 is a potential novel therapeutic option for lorlatinib-resistant ALK-rearranged lung cancer.

Abstract 6161: HER3 expression is associated with cold tumor immune microenvironment and the regulation of its signal in humanized breast cancer patient derived xenograft models converts to hot tumor

Abstract Background: HER3 signal pathways in cancer cells are thought to be involved in up-regulation of PD-L1 expression, resulting in “cold” tumor immune microenvironment (TIME). However, little is known on the association of HER3 expression and immune related molecular network in breast cancer and the functional role of HER3 signals in antitumor immune responses. Methods: The dataset of the TCGA study was analyzed to determine the correlation of HER3 expression and antitumor immune molecules. Two breast cancer patient-derived xenograft (PDX) models were treated with patritumab (anti-HER3 antibody), polyclonal activated autologous T cells (PATCs) or a combination of patritumab and PATCs (P-PATCs) in order to test immunological modulation by the regulation of HER3 signals. RT-PCR, immunohistochemical staining and CyTOF were performed in tumors or liver tissue for CD3, CD4, CD8a, CD137, CD134/OX40, CD80, IFN-γ and PD-L1, LAG3, TIM3, FOXP3. In addition, HER3-knockdown human breast cancer cell lines and PBMC were co-cultured and comprehensive cytokine expression in supernatant was analyzed by bioplex. Results: In TCGA dataset analysis, HER3 expression was inversely corelated with expression of CD4, CD8A, IFN-γ, granzyme A/B. In humanized PDX models, although treatment of PATCs or patritumab alone showed limited anti-tumor effects, P-PATCs treatment showed a significantly greater anti-tumor response. Interestingly, the proportion of CD137 expressing T cell infiltration and mRNA expression of CD137 was significantly higher in P-PATCs group compared to patritumab or the PATCs group, while the proportion of CD4/8 T cell infiltration was also higher in the P-PATCs group. CyTOF revealed that the proportion of CD137 and CD134/OX40 expressing cells was higher in P-PATCs group compared to the PATCs group. In co-culture of PBMC with HER3 knockdown cancer cells, the secretion level of immune stimulatory factors (IL2, IFN-g, TNF-a) increased and immune inhibitors (IL6, IL10) decreases. Conclusions: These data suggest that HER3 expression in breast cancer associated with “cold” TIME and the regulation of its signal converts “cold” TIME to “hot”. Thus, inhibition of HER3 signal might be of interesting concept for both regulation of oncologic signal and immunological modulation in HER3-expressing breast cancer. Further HER3 related immune modulation exploration in breast cancer treatment strategy needs to be clarified in the future. Citation Format: Sunao Tanaka, Eiji Suzuki, Takeshi Kotake, Shinpei Kawaoka, Kosuke Kawaguchi, Tomomi Nishimura, Fengling Pu, Masakazu Toi. HER3 expression is associated with cold tumor immune microenvironment and the regulation of its signal in humanized breast cancer patient derived xenograft models converts to hot tumor [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 6161.

Abstract 2612: Depleting EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer

Abstract Background & Goals: Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, is a major therapeutic target in several types of cancers. Several EGFR inhibitors are used to treat EGFR-positive colorectal cancer (CRC). Unfortunately, primary and acquired resistance to the inhibitors is very common. Current approaches to combating the resistance include combining EGFR inhibitors with agent(s) that targets compensatory signaling molecules (HER2, HER3, IGF1R, MET, KRAS, BRAF and PIK3CA). However, responses to such combination treatments are modest, if any. Overcoming drug resistance remains a major unmet medical need in EGFR-positive CRC. Results: Here, we show the main reason for CRC resistance to EGFR inhibitors and a therapeutic strategy that overcomes it. The sensitivity of CRC cells to EGFR inhibitors correlates with EGFR downregulation. PEPDG278D, a recombinant human protein that induces the degradation of both EGFR and HER2, strongly inhibits oncogenic signaling and growth of CRC in vitro and in vivo (subcutaneous tumors, orthotopic tumors, and patient-derived xenografts), regardless of activating mutations of KRAS, BRAF and PIK3CA. siRNA knockdown of EGFR or HER2 also inhibits CRC cells resistant to EGFR inhibitors. PEPDG278D indirectly suppresses HER3, IGF1R and MET by disrupting their heterodimerization with EGFR or HER2. While excessive tumor-generated EGFR ligands may block target engagement by PEPDG278D, inhibiting ligand shedding by aderbasib enables PEPDG278D to exert strong antitumor activity, and fluorouracil further enhances tumor inhibition. Collectively, our study shows that CRC resistance to EGFR inhibitors result primarily from their inability to eliminate their target and a PEPDG278D-centered combination treatment overcomes the resistance. Conclusions & Therapeutic Relevance: The therapeutic activity of PEPDG278D highlights that EGFR and HER2 remain indispensable to the survival of CRC cells resistant to EGFR inhibitors. The strong therapeutic activity of PEPDG278D-centered combination treatment in preclinical models of CRC, which carry clinically relevant gene mutations that confer resistance to current EGFR inhibitors, provides the scientific premise for potential evaluation of this recombinant human protein in patients with drug-resistant CRC. Citation Format: Lu Yang, Arup Bhattacharya, Yun Li, Sandra Sexton, Xiang Ling, Fengzhi Li, Yuesheng Zhang. Depleting EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal 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 2612.

Abstract 3272: Attenuation of HER3-EGFR signaling augments antitumor activity of panobinostat in triple negative breast cancer

Abstract Introduction: Histone deacetylases (HDACs) are dysregulated in human cancers, making them a valuable therapeutic target. The pan-HDAC inhibitor (HDACi) panobinostat is an approved anticancer drug for patients with multiple myeloma. It has been shown that triple negative breast cancer (TNBC), as compared to other breast cancer (BC) subtypes, is more sensitive to panobinostat. Despite the positive data observed in experimental models, to date this HDACi (panobinostat) has not yielded promising results in clinical trials for most solid tumors, including BC. Thus, a better understanding of the molecular mechanisms underlying the action of panobinostat is required to design effective therapeutic strategies against TNBC. Methods: Cell growth MTS assays and LIVE/DEAD cell viability assays were used to determine cell viability. Co-immunoprecipitation (Co-IP) and Western blot were performed to assess the expression, interaction and activation of proteins. Quantitative real-time PCR (qRT-PCR) assays were carried out to measure the expression levels of mRNAs. Lentiviral vectors containing cDNA or specific shRNAs were used to overexpress or knockdown gene expression. Tumor xenograft models via subcutaneous inoculation of MDA-MB-231 cells into nude mice were established to test the in vivo antitumor activity of panobinostat alone or its combination with the EGFR inhibitor gefitinib. Results: Elevated expression of HDAC1/2/3 was observed in BC and significantly associated with poor prognosis in BC patients. In general, all TNBC cells were sensitive to panobinostat-induced growth inhibition and apoptosis. However, in the TNBC cells with HER3-low/undetectable expression, treatment with panobinostat induced upregulation of HER3, and increased the levels of phosphorylated HER3 (p-HER3) and Akt (p-Akt). Specific knockdown of HER3 via shRNAs or inhibition of HER3 by our monoclonal antibody (4A7) sensitized TNBC cells to panobinostat-induced growth inhibition and apoptosis. Further studies revealed an enhanced interaction between HER3 and EGFR due to panobinostat-induced upregulation of HER3. Combinations of panobinostat and the EGFR inhibitor gefitinib exhibited a synergistic effect inhibiting cell growth and promoting apoptosis in TNBC cells. Moreover, in the tumor xenograft models established with MDA-MB-231 cells, panobinostat in combination with gefitinib significantly inhibited tumor growth and induced apoptosis in vivo. Conclusion: While panobinostat exhibits potent anti-proliferative/anti-survival activity in TNBC cells, upregulating HER3 may be an adaptive response compromising the therapeutic potential of panobinostat. Simultaneous targeting of HER3 and its oncogenic dimerization partner, such as EGFR holds promise to combat the compensatory signaling-initiated by HER3. This new therapeutic strategy potentially improves the outcomes of TNBC patients. Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Bolin Liu. Attenuation of HER3-EGFR signaling augments antitumor activity of panobinostat in triple negative 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 3272.

Abstract 397: Mesenchymal stem cell-derived systemic NRG-1 mediates a targetable molecular switch in metastatic prostate cancer

Abstract NRG-1 signals through epidermal growth factor receptor (EGFR) family members. Despite promising preclinical data, HER2 blockade in prostate cancer has failed to improve outcomes in clinical trials.1-4 We have discovered a novel complex systemic role of NRG-1 that drives sensitivity and resistance to androgen deprivation therapy (ADT) in castration-sensitive (CSPC) and castration resistant prostate cancer (CRPC), respectively. Significantly, sensitivity to ADT can be restored in CRPC by manipulating NRG-1 signaling. Our data posit NRG-1 as a systemic “switch” in prostate cancer. Plasma NRG-1 is elevated in our cohort of patients with metastatic CRPC (mean 4.9ng/ml) versus healthy controls (mean 3.2ng/ml, p<0.0001) or metastatic CSPC (mean 3.4ng/ml, p<0.0001). Paradoxically, high systemic NRG-1 predicted superior OS and PFS in CSPC but inferior clinical outcomes in CRPC. Consistently, opposite cell survival between LNCaP (CSPC) vs Du145 & PC3 (CRPC) was observed with recombinant human NRG-1 and enzalutamide (ENZ). ENZ combined with increasing NRG-1 concentration induced LNCaP cell death in an additive manner, but NRG-1 rescued Du145 & PC3 from ENZ-induced death in a dose-dependent manner. We next hypothesized that differential NRG-1 downstream signaling is mediated by engagement with distinct EGFR receptor dimers. While HER2 expression is similar, LNCaP express higher HER3 than Du145 & PC3. High ratio of HER2 to HER3 in Du145 & PC3 mirrors clinical reports of HER2 upregulation in CRPC5,6 and also predicts patient survival in the Stand Up to Cancer cohort.7 Significantly, we recapitulated the CRPC phenotype in LNCaP by inducing HER2 overexpression, HER3 suppression, and chemically-mediated HER2/HER3 dimerization. Conversely, HER2 knockdown sensitized Du145 cells to ENZ. Next, we queried composite single-cell whole human8 and CRPC9 transcriptomes for NRG-1 expression and discovered that the source of NRG-1 is the patients’ own mesenchymal stem cells (MSCs). Bone-derived MSC outgrowth cells produced copious NRG-1, which was increased substantially by coincubation with prostate cancer cell supernatants. Importantly, we were able to manipulate MSCs to reduce their NRG-1 production as a possible clinical translation. Similarly, patients treated with these compounds showed significant reduction in plasma NRG-1 levels after treatment. Additional clinical studies are pending. Together, our observations confirm that NRG-1 has differential signaling: HER3 homodimerization leads to prostate cancer cell death, whereas heterodimerization of HER3 with HER2 leads to prostate cancer cell survival via ADT resistance in a plasma NRG-1 dependent manner. Consequently, HER2 and HER3 receptor availability determine the molecular switch of NRG-1 in prostate cancer. Therefore, NRG-1 can be both a prognostic marker and therapeutic target in CRPC, and multiple clinical trials are pending. Citation Format: Jacob Orme, Roman Thaler, Dejie Wang, Siven Chinniah, Jacob Hirdler, Fernando Quevedo, Lance Pagliaro, Kwon Eugene, Alan Bryce, Brian Costello, Haojie Huang, Sean Park. Mesenchymal stem cell-derived systemic NRG-1 mediates a targetable molecular switch in metastatic prostate 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 397.

Depleting receptor tyrosine kinases EGFR and HER2 overcomes resistance to EGFR inhibitors in colorectal cancer

BackgroundEpidermal growth factor receptor (EGFR) inhibitors, including cetuximab and panitumumab, are valuable therapeutics for colorectal cancer (CRC), but resistance to these inhibitors is common. The reason for such resistance is not well understood, which hampers development of better therapeutic strategies. Although activating mutations in KRAS, BRAF and PIK3CA are considered major drivers of CRC resistance to EGFR inhibitors, therapeutic targeting of these drug resistance drivers has not produced substantial clinical benefit.MethodsWe exploited cell lines and mouse tumor models (cell line xenografts and patient derived xenografts) for experiments of genetic and pharmacologic depletion of EGFR and/or its family member HER2, including EGFR mutants, inhibition of EGFR ligand shedding, and biochemical analysis of signaling proteins, to delineate the mechanism of CRC resistance to EGFR inhibitors and to assess the therapeutic activity of PEPDG278D, which is a recombinant human protein that induces the degradation of both EGFR and HER2.ResultsThe sensitivity of CRC cells to cetuximab and panitumumab correlates with the ability of these drugs to induce EGFR downregulation. PEPDG278D strongly inhibits oncogenic signaling and growth of CRC cells by causing profound depletion of EGFR and HER2, regardless of activating mutations of KRAS, BRAF and PIK3CA. siRNA knockdown of EGFR or HER2 also inhibits CRC cells resistant to EGFR inhibitors. Tumors harboring mutated KRAS, BRAF and/or PIK3CA also overexpress EGFR ligands, further suggesting that EGFR signaling remains important to the tumors. While excessive tumor-generated high-affinity EGFR ligands block target engagement by PEPDG278D, aderbasib, an inhibitor of ADAM10 and ADAM17, enables PEPDG278D to exert strong antitumor activity by inhibiting ligand shedding. Moreover, adding fluorouracil, which is commonly used in CRC treatment, to the combination of PEPDG278D and aderbasib further enhances tumor inhibition.ConclusionsOur study shows that CRC resistance to EGFR inhibitors results primarily from the inability of the inhibitors to downregulate their target and that a PEPDG278D-based combination treatment overcomes the resistance.

HER2 promotes epithelial-mesenchymal transition through regulating osteopontin in gastric cancer

AimsHER2 and osteopontin (OPN) are both important biomarkers in gastric cancer (GC). The relationships between them remain to be revealed. The purpose of this study is to explore the role of OPN in epithelial-mesenchymal transition (EMT) in HER2 positive GCs. MethodsNanostring analysis was used to compare the mRNA levels of 730 cancer related genes between paired HER2 3+ and non-3+ areas in GC patients. Immunohistochemistry (IHC) staining was performed to analyze the expression levels of OPN, as well as EMT markers including E-cad, N-cad, twist and vimentin in both areas. To further verify the role of OPN in EMT, the expression levels of OPN and EMT markers, tumor invasion/migration were analyzed after down-regulating HER2 and OPN in GC cell lines MKN-45 and N-87. ResultsNanostring analysis identified 8 differential expression genes between HER2 3+ and non-3+ areas. Among them, the expression level of OPN was positively correlated with that of HER2. In GC specimens, OPN showed higher expression level in HER2 3+ areas where higher E-cad expression levels and lower N-cad and twist levels were also found. After knocking down OPN and HER2 by siRNA, both cell lines show decreased invasion/migration abilities, along with the down-regulation of the EMT phenotype, supporting by the decrease of E-cad, and the increase of N-cad and twist at both mRNA and protein levels. In addition, HER2 knock-down lead to a dramatic decrease of OPN expression. ConclusionsThese findings indicate that HER2 may promote EMT via the regulation of OPN in GCs.

HER3 PET Imaging Identifies Dynamic Changes in HER3 in Response to HER2 Inhibition with Lapatinib.

Standard therapy for HER2+ breast cancers includes HER2 inhibition. While HER2 inhibitors have significantly improved therapeutic outcomes, many patients remain resistant to therapy. An important intrinsic resistance mechanism to HER2 inhibition in some breast cancers is dynamic upregulation of HER3. Increase in HER3 expression that occurs in response to HER2 inhibition allows for continued growth signaling through HER2/HER3 heterodimers, promoting tumor escape. We hypothesized that a non-invasive method to image changes in HER3 expression would be valuable to identify those breast cancers that dynamically upregulate HER3 in response to HER2 inhibition. We further hypothesized that this imaging method could identify those tumors that would benefit by additional HER3 knockdown. In a panel of HER2+ breast cancer cell lines treated with the HER2 inhibitor lapatinib, we evaluate changes in HER3 expression and viability. Mouse HER2+ breast cancer models treated with lapatinib were imaged with a peptide-based HER3-specific PET imaging agent [68Ga]HER3P1 to assess for dynamic changes in tumoral HER3 expression and uptake confirmed by biodistribution. Subsequently, HER2+ cell lines were treated with the HER2 inhibitor lapatinib as well HER3-specific siRNA to assess for changes in viability and correlate with HER3 expression upregulation. For all statistical comparisons, P<0.05 was considered statistically significant. Lapatinib treatment of a panel of HER2+ breast cancer cell lines increased HER3 expression in the lapatinib-resistant cell line MDA-MB 453 but not the lapatinib-resistant cell-line HCC-1569. Evaluation of [68Ga]HER3P1 uptake in mice implanted with the HER2+ breast cancer cell lines MDA-MB453 or HCC-1569 prior to and after treatment with lapatinib demonstrated a significant increase in MDA-MB453 tumors only, consistent with in vitro findings. The additional knockdown of HER3 increased therapeutic efficacy of lapatinib only in MDA-MB453 cells, but not in HCC-1569 cells. HER3 PET imaging can be used to visualize dynamic changes in HER3 expression that occur in HER2+ breast cancers with HER2 inhibitor treatment and identify those likely to benefit by the addition of combination HER3 and HER2 inhibition.

Inhibition of HER Receptors Reveals Distinct Mechanisms of Compensatory Upregulation of Other HER Family Members: Basis for Acquired Resistance and for Combination Therapy.

Overexpression of members of the HER/erbB transmembrane tyrosine kinase family like HER2/erbB2/neu is associated with various cancers. Some heterodimers, especially HER2/HER3 heterodimers, are particularly potent inducers of oncogenic signaling. Still, from a clinical viewpoint their inhibition has yielded only moderate success so far, despite promising data from cell cultures. This suggests acquired resistance upon inhibitor therapy as one putative issue, requiring further studies in cell culture also aiming at rational combination therapies. In this paper, we demonstrate in ovarian carcinoma cells that the RNAi-mediated single knockdown of HER2 or HER3 leads to the rapid counter-upregulation of the respective other HER family member, thus providing a rational basis for combinatorial inhibition. Concomitantly, combined knockdown of HER2/HER3 exerts stronger anti-tumor effects as compared to single inhibition. In a tumor cell line xenograft mouse model, therapeutic intervention with nanoscale complexes based on polyethylenimine (PEI) for siRNA delivery, again reveals HER3 upregulation upon HER2 single knockdown and a therapeutic benefit from combination therapy. On the mechanistic side, we demonstrate that HER2 knockdown or inhibition reduces miR-143 levels with subsequent de-repression of HER3 expression, and validates HER3 as a direct target of miR-143. HER3 knockdown or inhibition, in turn, increases HER2 expression through the upregulation of the transcriptional regulator SATB1. These counter-upregulation processes of HER family members are thus based on distinct molecular mechanisms and may provide the basis for the rational combination of inhibitors.

Abstract LB-326: The role of HER3 in HER2-amplified cancers other than breast cancers

Abstract Introduction: Although recent studies showed that HER3 plays a key role in the pathophysiology of HER2-amplified breast cancers, its role in other cancers driven by HER2 amplification remains unknown. This study aimed to define the importance of HER3 signaling in other types of HER2-amplified cancer. Method: The expression and signaling activity of HER2, HER3, and downstream pathway proteins were studied in cell panels representing HER2-amplified cancers of the breast, bladder, colon and rectal, stomach, esophagus, lung, tongue, and endometrium along with controls lacking HER2 amplification. The time-dependent compensatory upregulation of HER3 in response to HER2 inhibitor therapy was assayed by western blotting and the apoptotic effects of HER2 inhibition were assayed by flow cytometry. The functional significance of HER3 in promoting tumorigenic growth in xenograft assays was determined in selected HER2-amplified cancer cell types by shRNA knockdown. Results: Many HER2-amplified cancers of the breast (HCC1569, HCC1954, HCC1419, UACC732, SUM190), bladder (CLS-439), stomach (N87, OE19), esophagus (TE4, KYSE410), lung (Calu3), and endometrium (USPC-ARC1, USPC-ARC2) showed HER3 activation, whereas HER2-nonamplified cancers of the same organs did not. With some exceptions, all HER2-amplified cancer cells undergo apoptosis when HER2 signaling is inhibited by lapatinib. Lapatinib induced apoptosis correlated with HER3 activation. Tumor growth was reduced upon HER3 knockdown in HER2-amplified cancers other than breast cancers, particularly cancers with constitutive HER3 phosphorylation. Significance: These results suggest that HER3 plays a functionally significant role in HER2 amplified cancers other than breast cancers, and may underlie resistance to HER2-targeting in these cancers. Combined HER2-HER3 targeting may be of value across the spectrum of different types of cancers. Citation Format: Avisek Majumder, Veronica Steri, Fernando Salangsang, Mark Moasser. The role of HER3 in HER2-amplified cancers other than breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-326.

Abstract LB-386: Novel multimodal precision immunotherapy by co-targeting the HER3 oncogenic signaling circuitry and PD-1 for head and neck squamous cell carcinoma

Abstract In prior studies, we have shown that persistent activation of the PI3K/mTOR signaling circuitry is the most frequently dysregulated signaling pathway in HNSCC (&amp;gt;80% of all HPV- and HPV+ cases), and that mTOR inhibitors (mTORi) exert potent antitumor activity in multiple experimental HNSCC model systems and in a recent Phase 2 clinical trial. In this regard, the overreliance on PI3K/mTOR signaling for tumor growth may in turn expose a cancer vulnerability that can be exploited therapeutically. However, the immunosuppressive effects of mTORi limit the potential benefits of their combination with new immune oncology (IO) agents. These include immune check point blockade with pembrolizumab and nivolumab (anti-PD-1), which have recently demonstrated anti-tumor activity in HNSCC, albeit only &amp;lt;20% of the patients show durable responses. There is urgent need to search for alternative approaches for HNSCC treatment. We conducted a kinome-wide siRNA screen to identify new therapeutic targets for HNSCC. This revealed that the ERBB3 gene, encoding HER3, is among the top 20 kinases whose knockdown (KD) decreases HNSCC cell proliferation. A counter screen analysis revealed that ERBB3 was the gene whose KD results in the highest reduction of mTOR pathway activation (indicated by pS6). Remarkably, HER3 KD specifically reduced activation of mTOR but not ERK in cells that do not harbor PIK3CA mutations, and immunoprecipitation of HER3 showed direct binding between HER3 and PI3K. Furthermore, a blocking antibody targeting HER3 (CDX-3379) potently inhibited the tumor growth of PIK3CA wild type but not PIK3CA mutant HNSCC cells. CDX-3379 also showed anti-tumor activity in 4MOSC1, our recently reported tobacco-associated syngeneic HNSCC model. Of importance, we found that HER3 is not expressed in immune cells, but CDX-3379 treatment led to a rapid increase in the accumulation of pro-immunogenic IL-2 and IL-7, concomitant with a reduction in multiple pro-tumorigenic and immune suppressive cytokines (e.g., IL-10, VEGF, G-CSF, GM-CSF, IL-6). This provided a rationale to combine HER3i with IO agents. Indeed, the combination of HER3 and PD-1 blockade elicited a remarkable beneficial effect, with an increase of tumor-infiltrating CD8+ T cells and ~70% of the mice exhibiting complete and durable (&amp;gt;6 months) responses and consequently a significant increase in overall survival. Overall, our findings suggest that persistent tyrosine phosphorylation of HER3 underlies aberrant PI3K/mTOR signaling in HNSCC harboring wild type PIK3CA, and that targeting HER3 may exert its anti-tumor effect by both reducing cancer-driving mTOR activity and reversing an immune evasive tumor microenvironment. These findings also support that co-targeting the HER3 signaling circuitry combined with PD-1 blockade may represent a novel multimodal precision therapeutic approach for HNSCC aimed at achieving durable responses and cancer remission. Citation Format: Zhiyong Wang, Yusuke Goto, Mara Gilardi, Michael Allevato, Victoria Wu, Robert Saddawi-konefka, Diego Alvarado, Alfredo Molinolo, J. Silvio Gutkind. Novel multimodal precision immunotherapy by co-targeting the HER3 oncogenic signaling circuitry and PD-1 for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-386.

Targeting human epidermal growth factor receptor 2 enhances radiosensitivity and reduces the metastatic potential of Lewis lung carcinoma cells

BackgroundSublethal radiation induces matrix metalloproteinase 9 (MMP-9)-mediated radioresistance in Lewis lung carcinoma (LLC) cells and their metastatic dissemination. We aim to determine if EGFR/HER2 activation associates with MMP-9-mediated radioresistance and invasiveness in irradiated LLC cells.MethodsLLC cells were treated with erlotinib or afatinib followed by sublethal radiation. After irradiation, we examined the phosphorylation of EGFR/HER2 and MMP-9 expression. Colony formation assay determined if the kinase inhibitors sensitize LLC cells to radiation. Matrigel-coated Boyden chamber assay assessed cellular invasiveness. Resulting tumors of wild-type LLC cells or HER2 knock-down mutant cells were irradiated to induce pulmonary metastases.ResultsAfatinib more effectively sensitized LLC cells to radiation and decreased invasiveness by inhibiting phosphorylation of EGFR, HER2, Akt, ERK, and p38, and down-regulating MMP-9 when compared to erlotinib. Afatinib abolished radiation-induced lung metastases in vivo. Furthermore, LLC HER2 knock-down cells treated with radiation had growth inhibition.ConclusionDual inhibition of radiation-activated EGFR and HER2 signaling by afatinib suppressed the proliferation and invasion of irradiated LLC cells. Increased radiosensitivity and decreased metastatic dissemination were observed by pharmacological or genetic HER2 inhibition in vivo. These findings indicate that HER2 plays a pivotal role in enhancing radioresistance and reducing metastatic potential of LLC cells.