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Trastuzumab therapy in HER2+ breast cancer patients has mixed success owing to acquired resistance to therapy. A detailed understanding of downstream molecular cascades resulting from trastuzumab resistance is yet to emerge. In this study, we investigate the cellular mechanisms underlying acquired resistance using trastuzumab-sensitive and -resistant cancer cells (BT474 and BT474R) treated with endogenous ligands EGF and HRG across time. We probe early receptor organization through microscopy and signaling events through multiomics measurements and assess the bioenergetic state through mitochondrial measurements. Integrative analyses of our measurements reveal significant alterations in EGF-treated BT474 HER2 membrane dynamics and robust downstream activation of PI3K/AKT/mTORC1 signaling. EGF-treated BT474R shows a sustained interferon-independent activation of the IRF1/STAT1 cascade, potentially contributing to trastuzumab resistance. Both cell lines exhibit temporally divergent metabolic demands and HIF1A-mediated stress responses. BT474R demonstrates inherently increased mitochondrial activity. HRG treatment in BT474R leads to a pronounced reduction in AR expression, affecting downstream lipid metabolism with implications for treatment response. Our results provide novel insights into mechanistic changes underlying ligand treatment in BT474 and BT474R and emphasize the pivotal role of endogenous ligands. These results can serve as a framework for furthering the understanding of trastuzumab resistance, with therapeutic implications for women with acquired resistance.

Introduction: Trastuzumab is commonly used to treat human epidermal growth factor receptor-2-positive (HER2+) breast cancer, but its efficacy is often limited by chemotherapy resistance. Recent studies have indicated that long non-coding RNAs (lncRNAs) play important roles in tumor progression and response to therapy. However, the regulatory mechanisms associating lncRNAs and trastuzumab resistance remain unknown. Methods: Quantitative polymerase chain reaction was performed to detect the expression of related genes. Western blot and immunofluorescence assays were used to evaluate protein expression levels. A series of gain- or loss-of-function assays confirmed the function of AGAP2-AS1 in trastuzumab resistance, both in vitro and in vivo. RNA immunoprecipitation and pull-down analyses were conducted to verify the interaction between METTL3/YTHDF2 and lncRNA AGAP2-AS1. Results: AGAP2-AS1 was upregulated in trastuzumab-resistant cells and SKBR-3R-generated xenografts in nude mice. Silencing AGAP2-AS1 significantly decreased trastuzumab-induced cytotoxicity both in vitro and in vivo. Furthermore, m6A methylation of AGAP2-AS1 was reduced in trastuzumab-resistant cells compared to that in parental cells. In addition, METTL3 increased m6A methylation of AGAP2-AS1, which finally induced the suppressed AGAP2-AS1 expression. Moreover, YTHDF2 was essential for METTL3-mediated m6A methylation of AGAP2-AS1. Functionally, AGAP2-AS1 regulated trastuzumab resistance by inducing autophagy and increasing ATG5 expression. Conclusion: we demonstrated that METTL3/YTHDF2-mediated m6A methylation increased the expression of AGAP2-AS1, which could promote trastuzumab resistance in breast cancer. AGAP2-AS1 regulates trastuzumab resistance by inducing autophagy. Therefore, AGAP2-AS1 may be a promising predictive biomarker and therapeutic target in patients with breast cancer. [ABSTRACT FROM AUTHOR]

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography–mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation. [ABSTRACT FROM AUTHOR]

Simple Summary: HER2-positive breast cancer, treated with trastuzumab, often develops resistance within a year in about 50% of patients. This study identifies ITGβ3 as a key factor in promoting resistance by upregulating stem cell markers through the Notch signaling pathway. Combining trastuzumab with the integrin inhibitor cilengitide significantly reduces these markers, suggesting a potential therapeutic approach to overcoming resistance. HER2-positive breast cancer, characterised by overexpressed HER2 levels, is associated with aggressive tumour behaviour and poor prognosis. Trastuzumab is a standard treatment; however, approximately 50% of patients develop resistance within one year. This study investigates the role of ITGβ3 in promoting stemness and resistance in HER2-positive breast cancer cell lines (HCC1599 and SKBR3). The findings demonstrate that chronic exposure to trastuzumab upregulates stem cell markers (SOX2, OCT4, KLF4, NANOG, SALL4, ALDH, BMI1, Nestin, Musashi 1, TIM3, CXCR4). Given the documented role of RGD-binding integrins in drug resistance and stemness, we specifically investigated their impact on resistant cells. Overexpression of ITGβ3 enhances the expression of these stem cell markers, while silencing ITGβ3 reduces their expression, suggesting a major role for ITGβ3 in maintaining stemness and resistance. Further analysis reveals that ITGβ3 activates the Notch signalling pathway, known for regulating stem cell maintenance. The combination of trastuzumab and cilengitide, an integrin inhibitor, significantly decreases the expression of stem cell markers in resistant cells, indicating a potential therapeutic strategy to overcome resistance. These results identify the importance of ITGβ3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is a malignancy with poor prognosis and high heterogeneity. For HER2-positive, PD-L1 negative metastatic GC patients, chemotherapy plus trastuzumab is the first-line therapy. However, such patients soon acquired resistance to treatment, especially to trastuzumab during the treatment. Improving the therapeutic resistance of HER2-positive, PD-L1 negative metastatic GC is still a dilemma. We present the case of a metastatic GC patient with HER2-positive and PD-L1-negative expression who suffered progression after a short remission with trastuzumab plus chemotherapy. The patient exhibited strong heterogeneity in the primary and metastatic lesions. His resistance to trastuzumab was overcome after the addition of a PD-1 inhibitor, after which he received a durable response for more than 8 months. In HER2-positive, PD-L1-negative metastatic GC, the addition of PD-1 inhibitors after first-line chemotherapy and trastuzumab treatment resistance may be an option.

Breast cancer antiestrogen resistance 4 (BCAR4) has been suggested that can modulate cell behavior, resulting in tumorigenesis and chemoresistance. However, the underlying mechanisms of BCAR4 in trastuzumab resistance (TR) is still elusive. Here, we explored the function and the underlying mechanism of BCAR4 involving in TR. We found that BCAR4 is significantly upregulated in trastuzumab‐resistant BC cells. Knockdown of BCAR4 could sensitize the BC cells to trastuzumab and suppress epithelial–mesenchymal transition (EMT). Mechanically, BCAR4 promotes yes‐associated protein 1 (YAP1) expression by competitively sponging miR‐665, to activated TGF‐β signaling. Reciprocally, YAP1 could occupy the BCAR4 promoter to enhance its transcription, suggesting that there exists a positive feedback regulation between YAP1 and BCAR4. Targeting the BCAR4/miR‐665/YAP1 axis may provide a novel insight of therapeutic approaches for TR in BC. [ABSTRACT FROM AUTHOR]

Simple Summary: tRF-27 has been identified as upregulated in both naïve trastuzumab-resistant HER2-positive breast cancer cells and patient samples, positioning it as a potential biomarker. However, the underlying mechanisms of this upregulation remain unclear. This study proposes that the abnormal transcription of specific tRNA-Cys-GCA isodecoders in naïve trastuzumab-resistant cells might contribute to the accumulation of tRF-27. Additionally, the reduction in H3K27me3 modifications at these tRNA genes, attributed to imbalances in histone modification enzymes, appears to influence tRNA transcription alterations. Building upon these findings, our research demonstrates that the application of a demethylase inhibitor may offer a potential strategy to overcome trastuzumab resistance. tRNA-derived fragments (tRFs) play crucial roles in cancer progression. Among them, tRF-27 has been identified as a key factor in promoting naïve trastuzumab resistance in HER2-positive breast cancer. However, the origin of tRF-27 remains uncertain. In this study, we propose that the upregulated expression of specific cysteine tRNAs may lead to the increased accumulation of tRF-27 in trastuzumab-resistant JIMT1 cells. Mechanistically, the reduced inhibitory H3K27me3 modification at the promoter regions of tRF-27-related tRNA genes in JIMT1 cells, potentially resulting from decreased EZH2 and increased KDM6A activity, may be a critical factor stimulating the transcriptional activity of these tRNA genes. Our research offers fresh insights into the mechanisms underlying elevated tRF-27 levels in trastuzumab-resistant breast cancer cells and suggests potential strategies to mitigate trastuzumab resistance in clinical treatments. [ABSTRACT FROM AUTHOR]

HER2-targeted therapies, such as Trastuzumab (Tz), have significantly improved the clinical outcomes for patients with HER2+ breast cancer (BC). However, treatment resistance remains a major obstacle. To elucidate functional and metabolic changes associated with acquired resistance, we characterized protein profiles of BC Tz-responder spheroids (RSs) and non-responder spheroids (nRSs) by a proteomic approach. Three-dimensional cultures were generated from the HER2+ human mammary adenocarcinoma cell line BT-474 and a derived resistant cell line. Before and after a 15-day Tz treatment, samples of each condition were collected and analyzed by liquid chromatography–mass spectrometry. The analysis of differentially expressed proteins exhibited the deregulation of energetic metabolism and mitochondrial pathways. A down-regulation of carbohydrate metabolism and up-regulation of mitochondria organization proteins, the tricarboxylic acid cycle, and oxidative phosphorylation, were observed in nRSs. Of note, Complex I-related proteins were increased in this condition and the inhibition by metformin highlighted that their activity is necessary for nRS survival. Furthermore, a correlation analysis showed that overexpression of Complex I proteins NDUFA10 and NDUFS2 was associated with high clinical risk and worse survival for HER2+ BC patients. In conclusion, the non-responder phenotype identified here provides a signature of proteins and related pathways that could lead to therapeutic biomarker investigation.

HER2-positive breast cancer, characterised by overexpressed HER2 levels, is associated with aggressive tumour behaviour and poor prognosis. Trastuzumab is a standard treatment; however, approximately 50% of patients develop resistance within one year. This study investigates the role of ITGβ3 in promoting stemness and resistance in HER2-positive breast cancer cell lines (HCC1954 and SKBR3). The findings demonstrate that chronic exposure to trastuzumab upregulates stem cell markers (SOX2, OCT4, KLF4, NANOG, SALL4, ALDH, BMI1, Nestin, Musashi 1, TIM3, CXCR4). Given the documented role of RGD-binding integrins in drug resistance and stemness, we specifically investigated their impact on resistant cells. Overexpression of ITGβ3 enhances the expression of these stem cell markers, while silencing ITGβ3 reduces their expression, suggesting a major role for ITGβ3 in maintaining stemness and resistance. Further analysis reveals that ITGβ3 activates the Notch signalling pathway, known for regulating stem cell maintenance. The combination of trastuzumab and cilengitide, an integrin inhibitor, significantly decreases the expression of stem cell markers in resistant cells, indicating a potential therapeutic strategy to overcome resistance. These results identify the importance of ITGβ3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy.

Abstract Background Patients with human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer and primary resistance to trastuzumab have a poor clinical outcome and lack good evidence to inform clinical decision. This study investigated the efficacy and safety of pyrotinib plus capecitabine in this population. Methods This phase 2 trial was conducted at 16 sites in China. Patients received oral pyrotinib 400 mg once daily and capecitabine 1000 mg/m2 twice a day on days 1–14 of each 21-day cycle until disease progression or intolerable toxicity. The primary endpoint was investigator-assessed progression-free survival (PFS). Results Between June 2019 and September 2021, 100 patients were enrolled with a median age of 51 years (range, 24–69). All patients had been treated with trastuzumab and 21 (21.0%) patients had prior use of pertuzumab. As of August 31, 2022, the median follow-up duration was 20.1 months (range, 1.3–38.2). The median PFS was 11.8 months (95% confidence interval [CI], 8.4–15.1), which crossed the pre-specified efficacy boundary of 8.0 months. The objective response rate was 70.0% (70/100), with a median duration of response of 13.8 months (95% CI, 10.2–19.3). The disease control rate was 87.0% (87/100). The median overall survival was not reached. The most common grade ≥ 3 treatment-emergent adverse event was diarrhea (24 [24.0%]). No treatment-related deaths occurred. Conclusions Pyrotinib plus capecitabine can be considered to be a treatment option in HER2-positive advanced breast cancer patients who have shown primary resistance to trastuzumab. Even in the era of modern anti-HER2 treatments, this clinical setting warrants more investigations to meet unmet needs. Trial registration ClinicalTrials.gov, NCT04001621. Retrospectively registered on June 28, 2019.

Background: Trastuzumab resistance accounts for chemotherapy failure in gastric cancer patients in clinical practice. The significance of long non-coding RNAs (lncRNAs) in the maintenance of drug resistance in gastric cancer has been already underlined. Method: This study aimed to identify the specific role of lncRNA-ATB in gastric cancer progression and trastuzumab resistance. The downstream miRs of lncRNA-ATB and target genes of miRs were predicted by bioinformatics analysis and verified using dual luciferase reporter assay. Loss- and gain-function assays were performed to explore the roles of lncRNA-ATB, miR-200c, and zinc-finger protein 217 (ZNF217) in the cell functions and trastuzumab resistance of a trastuzumab-resistant gastric cancer cell line (NCI-N87-TR). Result: LncRNA-ATB was upregulated, while miR-200c was downregulated. Depletion of lncRNA-ATB or miR-200c elevation led to a decrease in malignant properties of NCI-N87-TR cells. LncRNA-ATB could negatively target miR- 200c, which in turn inversely targeted and reduced the expression of ZNF217. Silencing of ZNF217 could inhibit cell viability and migration. Conclusion: lncRNA-ATB promoted the progression and trastuzumab resistance of gastric cancer by repressing miR-200c via ZNF217 upregulation. [ABSTRACT FROM AUTHOR]

The developed resistance of trastuzumab remained a problem for clinical therapy of HER2-positive breast cancer. However, effects of YAP/TAZ pathway on resistance of trastuzumab have not been explored. Tumor tissues were collected from 40 breast cancer patients for clinical studies. For in vitro studies, human breast cancer cell lines SK-BR-3-TS was obtained, and trastuzumab resistant model SK-BR-3-TR was constructed. Cell viability was determined using MTT assay. Cell apoptosis was analyzed by flow cytometry. Protein and mRNA expression was measured using western blotting and RT-qPCR, respectively. The mRNA and protein level of YAP was significantly increased in the tumor tissues of HER2-positive breast cancer patients. Consistently, the expression of YAP and TAZ were both dramatically upregulated in SK-BR-3-TR cells. The cell viability was increased, while cell apoptosis was inhibited in SK-BR-3-TS cells compared with SK-BR-3-TS. The depletion of YAP by si-YAP reversed the YAP/TAZ expression, cell viability and cell apoptosis in SK-BR-3-TR cells. YAP/TAZ pathway might induce the trastuzumab resistance in HER2-positive breast cancer and targeting YAP would be an alternative way for the clinical therapeutic methods of HER2 positive breast cancer. [ABSTRACT FROM AUTHOR]

Introduction: HER2 overexpression induces cancer aggression and frequent recurrences in many solid tumors. Because HER2 overproduction is generally followed by gene amplification, inhibition of protein–protein interaction (PPI) between transcriptional factor ELF3 and its coactivator MED23 has been considered an effective but challenging strategy. Objectives: This study aimed to determine the hotspot of ELF3-MED23 PPI and further specify the essential residues and their key interactions in the hotspot which are controllable by small molecules with significant anticancer activity. Methods: Intensive biological evaluation methods including SEAP, fluorescence polarization, LC-MS/MS-based quantitative, biosensor, GST-pull down assays, and in silico structural analysis were performed to determine hotspot of ELF3-MED23 PPI and to elicit YK1, a novel small molecule PPI inhibitor. The effects of YK1 on possible PPIs of MED23 and the efficacy of trastuzumab were assessed using cell culture and tumor xenograft mouse models. Results: ELF3-MED23 PPI was found to be specifically dependent on H-bondings between D400, H449 of MED23 and W138, I140 of ELF3 for upregulating HER2 gene transcription. Employing YK1, we confirmed that interruption on these H-bondings significantly attenuated the HER2-mediated oncogenic signaling cascades and exhibited significant in vitro and in vivo anticancer activity against HER2-overexpressing breast and gastric cancers even in their trastuzumab refractory clones. Conclusion: Our approach to develop specific ELF3-MED23 PPI inhibitor without interfering other PPIs of MED23 can finally lead to successful development of a drug resistance-free compound to interrogate HER2 biology in diverse conditions of cancers overexpressing HER2.

Monoclonal antibody-based therapy has achieved great success and is now one of the most crucial therapeutic modalities for cancer therapy. The first monoclonal antibody authorized for treating human epidermal growth receptor 2 (HER2)-positive breast cancer is trastuzumab. However, resistance to trastuzumab therapy is frequently encountered and thus significantly restricts the therapeutic outcomes. To address this issue, tumor microenvironment (TME) pH-responsive nanoparticles (NPs) were herein developed for systemic mRNA delivery to reverse the trastuzumab resistance of breast cancer (BCa). This nanoplatform is comprised of a methoxyl-poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) copolymer with a TME pH-liable linker (Meo-PEG-Dlinkm-PLGA) and an amphiphilic cationic lipid that can complex PTEN mRNA via electrostatic interaction. When the long-circulating mRNA-loaded NPs build up in the tumor after being delivered intravenously, they could be efficiently internalized by tumor cells due to the TME pH-triggered PEG detachment from the NP surface. With the intracellular mRNA release to up-regulate PTEN expression, the constantly activated PI3K/Akt signaling pathway could be blocked in the trastuzumab-resistant BCa cells, thereby resulting in the reversal of trastuzumab resistance and effectively suppress the development of BCa.

Trastuzumab notably improves the outcome of human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients, however, resistance to trastuzumab remains a major hurdle to clinical treatment. In the present study, we identify a circular RNA intimately linked to trastuzumab resistance. circ-β-TrCP, derived from the back-splicing of β-TrCP exon 7 and 13, confers trastuzumab resistance by regulating NRF2-mediated antioxidant pathway in a KEAP1-independent manner. Concretely, circ-β-TrCP encodes a novel truncated 343-amino acid peptide located in the nucleus, referred as β-TrCP-343aa, which competitively binds to NRF2, blocks SCFβ-TrCP-mediated NRF2 proteasomal degradation, and this protective effect of β-TrCP-343aa on NRF2 protein requires GSK3 activity. Subsequently, the elevated NRF2 transcriptionally upregulates a cohort of antioxidant genes, giving rise to trastuzumab resistance. Moreover, the translation ability of circ-β-TrCP is inhibited by eIF3j under both basal and oxidative stress conditions, and eIF3j is transcriptionally repressed by NRF2, thus forming a positive feedback circuit between β-TrCP-343aa and NRF2, expediting trastuzumab resistance. Collectively, our data demonstrate that circ-β-TrCP-encoded β-TrCP protein isoform drives HER2-targeted therapy resistance in a NRF2-dependent manner, which provides potential therapeutic targets for overcoming trastuzumab resistance.

Background: Patients with human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer and primary resistance to trastuzumab have a poor clinical outcome and lack good evidence to inform clinical decision. This study investigated the efficacy and safety of pyrotinib plus capecitabine in this population. Methods: This phase 2 trial was conducted at 16 sites in China. Patients received oral pyrotinib 400 mg once daily and capecitabine 1000 mg/m2 twice a day on days 1–14 of each 21-day cycle until disease progression or intolerable toxicity. The primary endpoint was investigator-assessed progression-free survival (PFS). Results: Between June 2019 and September 2021, 100 patients were enrolled with a median age of 51 years (range, 24–69). All patients had been treated with trastuzumab and 21 (21.0%) patients had prior use of pertuzumab. As of August 31, 2022, the median follow-up duration was 20.1 months (range, 1.3–38.2). The median PFS was 11.8 months (95% confidence interval [CI], 8.4–15.1), which crossed the pre-specified efficacy boundary of 8.0 months. The objective response rate was 70.0% (70/100), with a median duration of response of 13.8 months (95% CI, 10.2–19.3). The disease control rate was 87.0% (87/100). The median overall survival was not reached. The most common grade ≥ 3 treatment-emergent adverse event was diarrhea (24 [24.0%]). No treatment-related deaths occurred. Conclusions: Pyrotinib plus capecitabine can be considered to be a treatment option in HER2-positive advanced breast cancer patients who have shown primary resistance to trastuzumab. Even in the era of modern anti-HER2 treatments, this clinical setting warrants more investigations to meet unmet needs. Trial registration: ClinicalTrials.gov, NCT04001621. Retrospectively registered on June 28, 2019. [ABSTRACT FROM AUTHOR]

Abstract Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is characterized by invasive growth, rapid metastasis and chemoresistance. Trastuzumab is an effective treatment for HER2+ breast cancer; however, trastuzumab resistance leads to cancer relapse and metastasis. CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) has been considered as a new immune checkpoint for tumor-induced immunosuppression. The role of CMTM6 in trastuzumab resistance remains unknown. Here, we uncover a role of CMTM6 in trastuzumab-resistant HER2+ breast cancer. CMTM6 expression was upregulated in trastuzumab-resistant HER2+ breast cancer cell. Patients with high CMTM6 expressing HER2+ breast cancer had worse overall and progression-free survival than those with low CMTM6 expression. In vitro, CMTM6 knockdown inhibited the proliferation and migration of HER2+ breast cancer cells, and promoted their apoptosis, while CMTM6 overexpression reversed these effects. CMTM6 and HER2 proteins were co-localized on the surface of breast cancer cells, and CMTM6 silencing reduced HER2 protein levels in breast cancer cells. Co-immunoprecipitation revealed that CMTM6 directly interacted with HER2 in HER2+ breast cancer cells, and CMTM6 overexpression inhibited HER2 ubiquitination. Collectively, these findings highlight that CMTM6 stabilizes HER2 protein, contributing to trastuzumab resistance and implicate CMTM6 as a potential prognostic marker and therapeutic target for overcoming trastuzumab resistance in HER2+ breast cancer.

Abstract Background The emergence of de novo or intrinsic trastuzumab resistance is exceedingly high in breast cancer that is HER2 positive and correlates with an abundant cancer stem cell (CSC)-like population. We sought to examine the capacity of β-escin, an anti-inflammatory drug, to address trastuzumab resistance in HER2-positive breast cancer cells. Methods The effect of β-escin on trastuzumab-resistant and -sensitive cell lines in vitro was evaluated for apoptosis, expression of HER2 family members, and impact on CSC-like properties. An in vivo model of trastuzumab-resistant JIMT-1 was used to examine the efficacy and toxicity of β-escin. Results β-escin induced mitochondrial-mediated apoptosis accompanied by reactive oxygen species (ROS) production and increased active p18Bax fragmentation, leading to caspase-3/-7 activation. Attenuation of CSC-related features by β-escin challenge was accompanied by marked reductions in CD44high/CD24low stem-like cells and aldehyde dehydrogenase 1 (ALDH1) activity as well as hindrance of mammosphere formation. β-escin administration also significantly retarded tumor growth and angiogenesis in a trastuzumab-resistant JIMT-1 xenograft model via downregulation of CSC-associated markers and intracellular domain HER2. Importantly, β-escin selectively inhibited malignant cells and was less toxic to normal mammary cells, and no toxic effects were found in liver and kidney function in animals. Conclusions Taken together, our findings highlight β-escin as a promising candidate for the treatment of trastuzumab-resistant HER2-positive breast cancers.

Breast cancer is the most common cancer in women. The human epidermal growth factor receptor 2 (HER2) overexpressing subtype is related to poor prognosis with an aggressive phenotype and is reported as one of the most commonly seen subtypes. Trastuzumab is prevalently used as a treatment method for HER2+ breast cancer however, resistance to the drug frequently occurs following the treatment. MicroRNAs (miRNAs) are 19-23 nucleotide long small RNAs, which regulate gene expression at posttranscriptional level and studies show that there are differentially expressed miRNAs between drug sensitive and resistant groups, indicating that they might have some key roles in drug effectiveness. In this study, the aim is to find out the role of miR-216b-5p in trastuzumab resistance. SK-BR-3 cells developed resistance to trastuzumab after continuous treatment with increasing concentrations of the drug for 6 months. To investigate the effect of miR-216b-5p on cancer cell behavior in resistance state, proliferation, motility, and invasion capacities of these resistant cells were analyzed by xCELLigence real-time cell analyzer. To further understand the molecular mechanisms underlying the regulation of resistant SK-BR-3 cells by miR-216b-5p, microarray analysis was performed. Apoptosis analysis was also performed since the pathway enrichment analysis pointed out cell death related pathways. The proliferation, motility, and invasion capacities of the miR-216b-5p transfected resistant cells were diminished compared to sensitive cells. We identified the necroptosis signaling pathway as the result of microarray and pathway enrichment analyses. STAT1, IRF9, and PKR were validated as the significant elements of the pathway, which are also the putative targets of miR-216b-5p. Our apoptosis analysis showed that a significant amount of trastuzumab resistant SK-BR-3 cells entered to late apoptosis/necrosis stage upon miR-216b-5p overexpression, it could be concluded that reexpression of miR-216b-5p sensitizes trastuzumab resistance through necroptosis in breast cancer. [ABSTRACT FROM AUTHOR]

[Display omitted] • ELF3-MED23 hotspot was efficiently deciphered by using customized small molecules. • ELF3-MED23 PPI depends on specific H-bondings to upregulate HER2 expression. • PPI inhibitory activity of YK1 was specific to ELF3-MED23. • YK1-mediated specific H-bond interruption induces promising anticancer activities. • Anticancer effect of YK1 was even significant against trastuzumab-refractory clones. • YK1 held highly drug-like properties showing favorable PK profile. HER2 overexpression induces cancer aggression and frequent recurrences in many solid tumors. Because HER2 overproduction is generally followed by gene amplification, inhibition of protein–protein interaction (PPI) between transcriptional factor ELF3 and its coactivator MED23 has been considered an effective but challenging strategy. This study aimed to determine the hotspot of ELF3-MED23 PPI and further specify the essential residues and their key interactions in the hotspot which are controllable by small molecules with significant anticancer activity. Intensive biological evaluation methods including SEAP, fluorescence polarization, LC-MS/MS-based quantitative, biosensor, GST-pull down assays, and in silico structural analysis were performed to determine hotspot of ELF3-MED23 PPI and to elicit YK1, a novel small molecule PPI inhibitor. The effects of YK1 on possible PPIs of MED23 and the efficacy of trastuzumab were assessed using cell culture and tumor xenograft mouse models. ELF3-MED23 PPI was found to be specifically dependent on H-bondings between D400, H449 of MED23 and W138, I140 of ELF3 for upregulating HER2 gene transcription. Employing YK1, we confirmed that interruption on these H-bondings significantly attenuated the HER2-mediated oncogenic signaling cascades and exhibited significant in vitro and in vivo anticancer activity against HER2-overexpressing breast and gastric cancers even in their trastuzumab refractory clones. Our approach to develop specific ELF3-MED23 PPI inhibitor without interfering other PPIs of MED23 can finally lead to successful development of a drug resistance-free compound to interrogate HER2 biology in diverse conditions of cancers overexpressing HER2. [ABSTRACT FROM AUTHOR]

Purpose: Dysregulation of the PI3K pathway is one of the most common events in breast cancer. Here we investigate the activity of the PI3K inhibitor MEN1611 at both molecular and phenotypic levels by dissecting and comparing its profile and efficacy in HER2 + breast cancer models with other PI3K inhibitors. Methods: Models with different genetic backgrounds were used to investigate the pharmacological profile of MEN1611 against other PI3K inhibitors. In vitro studies evaluated cell viability, PI3K signaling, and cell death upon treatment with MEN1611. In vivo efficacy of the compound was investigated in cell line- and patient-derived xenografts models. Results: Consistent with its biochemical selectivity, MEN1611 demonstrated lower cytotoxic activity in a p110δ-driven cellular model when compared to taselisib, and higher cytotoxic activity in the p110β-driven cellular model when compared to alpelisib. Moreover, MEN1611 selectively decreased the p110α protein levels in PIK3CA mutated breast cancer cells in a concentration- and proteasome-dependent manner. In vivo, MEN1611 monotherapy showed significant and durable antitumor activity in several trastuzumab-resistant PIK3CA-mutant HER2 + PDX models. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. Conclusions: The profile of MEN1611 and its antitumoral activity suggest an improved profile as compared to pan-inhibitors, which are limited by a less than ideal safety profile, and isoform selective molecules, which may potentially promote development of resistance mechanisms. The compelling antitumor activity in combination with trastuzumab in HER2 + trastuzumab-resistant, PIK3CA mutated breast cancer models is at the basis of the ongoing B-Precise clinical trial (NCT03767335). [ABSTRACT FROM AUTHOR]

BACKGROUND: The albumin-bilirubin (ALBI) score is a novel indicator of liver function. Some studies showed that the ALBI score was a predictive marker for the prognosis and efficacy of drug therapy in malignancies. We aimed to assess the predicted role of ALBI score in the sensitivity to therapy with trastuzumab in patients with human epidermal growth factor receptor 2 (HER2) positive breast cancer (BC). The clinical data of 226 HER2-positive BC patients at the Harbin Medical University Cancer Hospital from January 2017 and December 2018 were retrospectively collected. The ALBI score was calculated with serum albumin and bilirubin before diagnosis. The associations between ALBI score and trastuzumab resistance were analyzed by logistic regression analyses. The patients with trastuzumab resistance had higher ALBI scores compared with the patients without trastuzumab resistance. Moreover, there were weak correlations between the ALBI score and lymph node status (P = 0.093). In addition, multivariate analysis revealed that the ALBI score was an independent prognostic factor for trastuzumab resistance in HER2-positive BC. High ALBI score is associated with trastuzumab resistance in HER2-positive BC. Future studies are needed. [ABSTRACT FROM AUTHOR]

Trastuzumab plus chemotherapy is the standard of care for the first-line treatment of patients with HER2+ advanced esophagogastric (EG) cancer. Nevertheless, patients frequently develop resistance. In preclinical models, we identified the overexpression of Fibroblast Growth Factor Receptor (FGFR) 3 as a mechanism potentially involved in trastuzumab-acquired resistance. FGFR inhibition could be a potential mechanism as a second-line treatment. In this Simon's two-stage phase 2, single arm study, patients with advanced EG cancer refractory to trastuzumab-containing therapies received pemigatinib, an inhibitor of FGFR. The primary end point was the 12-week progression-free survival rate. Translational analyses were performed on tissue and plasma samples. Eight patients were enrolled in the first stage. Although the 6-week disease control rate was 25%, only one patient achieved a stable disease after 12 weeks of treatment. The trial was discontinued before the second stage. Two out of six evaluable tumor samples expressed FGFR3. No FGFRs amplification was detected. HER2 amplification was lost in three out of eight patients. Three patients had an high Tumor Mutational Burden, and two of them are significantly long-term survivors. These results do not support the therapeutic efficacy of targeting FGFR in unselected patients with advanced EG cancer, who are refractory to trastuzumab-containing therapies. [ABSTRACT FROM AUTHOR]

Monoclonal antibody-based therapy has achieved great success and is now one of the most crucial therapeutic modalities for cancer therapy. The first monoclonal antibody authorized for treating human epidermal growth receptor 2 (HER2)-positive breast cancer is trastuzumab. However, resistance to trastuzumab therapy is frequently encountered and thus significantly restricts the therapeutic outcomes. To address this issue, tumor microenvironment (TME) pH-responsive nanoparticles (NPs) were herein developed for systemic mRNA delivery to reverse the trastuzumab resistance of breast cancer (BCa). This nanoplatform is comprised of a methoxyl-poly (ethylene glycol)- b -poly (lactic- co -glycolic acid) copolymer with a TME pH-liable linker (Meo-PEG- Dlink m -PLGA) and an amphiphilic cationic lipid that can complex PTEN mRNA via electrostatic interaction. When the long-circulating mRNA-loaded NPs build up in the tumor after being delivered intravenously, they could be efficiently internalized by tumor cells due to the TME pH-triggered PEG detachment from the NP surface. With the intracellular mRNA release to up-regulate PTEN expression, the constantly activated PI3K/Akt signaling pathway could be blocked in the trastuzumab-resistant BCa cells, thereby resulting in the reversal of trastuzumab resistance and effectively suppress the development of BCa. A tumor microenvironment (TME) pH-responsive nanoparticle (NP) platform was developed to in vivo transport PTEN mRNA for the reversal of trastuzumab resistance and effective breast cancer (BCa) therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Platelet-derived growth factor receptor (PDGFR) plays an essential role in the proliferation and invasion of malignant cancer cells. However, the functional role of PDGFR alpha (PDGFRA) in HER2-positive (HER2+) breast cancer has not been fully clarified yet. Thus, the objective of this study was to investigate the clinical significance of PDGFRA and the therapeutic potential of PDGFR inhibitors as part of an effort to overcome trastuzumab (TRZ) resistance. Aberrant PDGFRA expression is closely associated with decreased survival in HER2+ breast cancers. Therefore, we established BT474 trastuzumab-sensitive (TRZ_S) and trastuzumab-resistant (TRZ_R) cells to investigate the association between PDGFR signaling and TRZ resistance. We found that PDGFRA was significantly upregulated in the BT474 TRZ_R cells. In addition, IL-6 expression, which was also found to be upregulated in the TRZ_R cells, was induced by PDGFC, a ligand of PDGFR. Next, we investigated the effects of ponatinib and sunitinib, PDGFR inhibitors, on the BT474 TRZ_R and HCC1954 (TRZ-resistant cell line) cells. These inhibitors decreased cell viability and migration in a dose-dependent manner. Additionally, IL-6 expression was decreased by ponatinib in both the BT474 TRZ_R and HCC1954 cells. In contrast, IL-6 was not suppressed by TRZ, implying that the PDGFRA/STAT3/IL-6 axis is associated with resistance to TRZ. In addition, we found that STAT3 and ERK phosphorylation were increased in the BT474 TRZ_R cells. IL-6 expression was suppressed by a STAT3 inhibitor, indicating that IL-6 expression is modulated downstream of STAT3. Taken together, these results suggest that PDGFRA could serve as a therapeutic target to overcome TRZ resistance. [ABSTRACT FROM AUTHOR]

Abstract Purpose The aim of this study was to explore the role of galectin‐3 in human epidermal growth factor receptor 2 (HER2)‐positive breast cancer cells and the potential mechanism. Methods Kaplan–Meier (KM)‐plot and The Cancer Genome Atlas (TCGA) databases were used to study the role of galectin‐3 in the prognosis of HER2‐positive breast cancer. The effects of galectin‐3 on cell proliferation, migration, invasion, and colony formation ability in HER2‐positive breast cancer cells were examined. The relationship between galectin‐3 and important components in the HER2 pathways, including HER2, epidermal growth factor receptor (EGFR), protein kinase B (AKT), and phosphatase and tensin homolog (PTEN), was further studied. Lentivirus and CRISPR/Cas9 were used to construct stable cell lines. Cell counting kit‐8 (CCK‐8) and apoptosis assays were used to study the relationship between galectin‐3 and trastuzumab. The effect of galectin‐3 on cell stemness was studied by mammosphere formation assay. The effects of galectin‐3 on stemness biomarkers and the Notch1 pathway were examined. Tumorigenic models were used to evaluate the effects of galectin‐3 on tumorigenesis and the therapeutic effect of trastuzumab in vivo. Results HER2‐positive breast cancer patients with a high expression level of LGALS3 (the gene encoding galectin‐3) messenger RNA (mRNA) showed a poor prognosis. Galectin‐3 promoted cancer malignancy through phosphoinositide 3‐kinase (PI3K)/AKT signaling pathway activation and upregulated stemness by activating the Notch1 signaling pathway in HER2‐positive breast cancer cells. These two factors contributed to the enhancement of trastuzumab resistance in cells. Knockout of LGALS3 had a synergistic therapeutic effect with trastuzumab both in vitro and in vivo. Conclusions Galectin‐3 may represent a prognostic predictor and therapeutic target for HER2‐positive breast cancer.

Simple Summary: Cortactin (CTTN) is an actin-binding protein that is mainly known for its ability to promote cancer progression. It is unclear, however, whether CTTN affects tumor initiation and anti-tumor drug resistance in breast cancer. Here, we investigated the potential role of CTTN as a novel poor prognostic biomarker and possible therapeutic target of trastuzumab resistance for HER2 positive breast cancer. Our findings showed that CTTN enhances tumor initiation and increases anti-HER2 drug resistance by activating the DKK-1/Wnt/β-catenin signaling pathway. CTTN-induced cancer stem cell-like properties were reversed by treatment of β-catenin/TCF inhibitor. Furthermore, combinational treatment with trastuzumab and β-catenin/TCF inhibitor overcame CTTN-induced trastuzumab resistance. These results suggest that combined treatment of trastuzumab and Wnt signaling inhibitors could be an effective therapeutic strategy to treat HER2+ breast tumors expressing high levels of CTTN. Collectively, we suggest that CTTN could be a potential target for treatment of trastuzumab resistance in HER2 positive breast cancer patients. Background: Despite the therapeutic success of trastuzumab, HER2 positive (HER2+) breast cancer patients continue to face significant difficulties due to innate or acquired drug resistance. In this study we explored the potential role of CTTN in inducing trastuzumab resistance of HER2+ breast cancers. Methods: Genetic changes of CTTN and survival of HER2+ breast cancer patients were analyzed in multiple breast cancer patient cohorts (METABRIC, TCGA, Kaplan-Meier (KM) plotter, and Hanyang University cohort). The effect of CTTN on cancer stem cell activity was assessed using the tumorsphere formation, ALDEFLUOR assay, and by in vivo xenograft experiments. CTTN-induced trastuzumab resistance was assessed by the sulforhodamine B (SRB) assay, colony formation assays, and in vivo xenograft model. RNA-seq analysis was used to clarify the mechanism of trastuzumab resistance conferred by CTTN. Results: Survival analysis indicated that CTTN overexpression is related to a poor prognosis in HER2+ breast cancers (OS, p = 0.05 in the Hanyang University cohort; OS, p = 0.0014 in KM plotter; OS, p = 0.008 and DFS, p = 0.010 in METABRIC). CTTN overexpression-induced cancer stem cell-like characteristics in experiments of tumorsphere formation, ALDEFLUOR assays, and in vivo limiting dilution assays. CTTN overexpression resulted in trastuzumab resistance in SRB, colony formation assays, and in vivo xenograft models. Mechanistically, the mRNA and protein levels of DKK-1, a Wnt antagonist, were downregulated by CTTN. Treatment of the β-catenin/TCF inhibitor reversed CTTN-induced cancer stem cell-like properties in vitro. Combination treatment with trastuzumab and β-catenin/TCF inhibitor overcame trastuzumab resistance conferred by CTTN overexpression in in vitro colony formation assays. Conclusions: CTTN activates DKK-1/Wnt/β-catenin signaling to induce trastuzumab resistance. We propose that CTTN is a novel biomarker indicating a poor prognosis and a possible therapeutic target for overcoming trastuzumab resistance. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is characterized by invasive growth, rapid metastasis and chemoresistance. Trastuzumab is an effective treatment for HER2+ breast cancer; however, trastuzumab resistance leads to cancer relapse and metastasis. CKLF-like MARVEL transmembrane domain-containing 6 (CMTM6) has been considered as a new immune checkpoint for tumor-induced immunosuppression. The role of CMTM6 in trastuzumab resistance remains unknown. Here, we uncover a role of CMTM6 in trastuzumab-resistant HER2+ breast cancer. CMTM6 expression was upregulated in trastuzumab-resistant HER2+ breast cancer cell. Patients with high CMTM6 expressing HER2+ breast cancer had worse overall and progression-free survival than those with low CMTM6 expression. In vitro, CMTM6 knockdown inhibited the proliferation and migration of HER2+ breast cancer cells, and promoted their apoptosis, while CMTM6 overexpression reversed these effects. CMTM6 and HER2 proteins were co-localized on the surface of breast cancer cells, and CMTM6 silencing reduced HER2 protein levels in breast cancer cells. Co-immunoprecipitation revealed that CMTM6 directly interacted with HER2 in HER2+ breast cancer cells, and CMTM6 overexpression inhibited HER2 ubiquitination. Collectively, these findings highlight that CMTM6 stabilizes HER2 protein, contributing to trastuzumab resistance and implicate CMTM6 as a potential prognostic marker and therapeutic target for overcoming trastuzumab resistance in HER2+ breast cancer. [ABSTRACT FROM AUTHOR]

Resistance to targeted therapy is one of the critical obstacles in cancer management. Resistance to trastuzumab frequently develops in the treatment for HER2+ cancers. The role of protein tyrosine phosphatases (PTPs) in trastuzumab resistance is not well understood. In this study, we aim to identify pivotal PTPs affecting trastuzumab resistance and devise a novel counteracting strategy. Four public datasets were used to screen PTP candidates in relation to trastuzumab responsiveness in HER2+ breast cancer. Tyrosine kinase (TK) arrays were used to identify kinases that linked to protein tyrosine phosphate receptor type O (PTPRO)-enhanced trastuzumab sensitivity. The efficacy of small activating RNA (saRNA) in trastuzumab-conjugated silica nanoparticles was tested for PTPRO upregulation and resistance mitigation in cell models, a transgenic mouse model, and human cancer cell line-derived xenograft models. PTPRO was identified as the key PTP which influences trastuzumab responsiveness and patient survival. PTPRO de-phosphorated several TKs, including the previously overlooked substrate ERBB3, thereby inhibiting multiple oncogenic pathways associated with drug resistance. Notably, PTPRO, previously deemed "undruggable," was effectively upregulated by saRNA-loaded nanoparticles. The upregulated PTPRO simultaneously inhibited ERBB3, ERBB2, and downstream SRC signaling pathways, thereby counteracting trastuzumab resistance. Antibody-conjugated saRNA represents an innovative approach for targeting "undruggable" PTPs. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Activation of the PI3K/AKT/mTOR pathway in patients with HER2-positive breast cancer is associated with acquired resistance to trastuzumab. This randomized controlled trial (RCTs) meta-analysis was designed to evaluate the clinical efficacy and safety of PI3K/Akt/mTOR inhibitors in combination with trastuzumab in HER2-positive breast cancer. MATERIALS AND METHODS: We searched on Web of Knowledge, PubMed, Embase, Cochrane, CNKI, and ClinicalTrials. Gov for RCTs comparing PI3K/Akt/mTOR inhibitors plus trastuzumab vs. standard trastuzumab treatments. Pooled estimates of progression-free survival (PFS), pathologic complete response (pCR), and incidence of adverse events were determined. RESULTS: 5 studies out of 610 were found to be eligible and were included in our analysis (n=1,548 participants). PI3K/Akt/mTOR inhibitors combination with trastuzumab treatments resulted in a statistically significant increase in PFS compared with conventional trastuzumab therapy (HR 0.82; 95% CI: 0.76-0.90; p<0.00001). The new combination treatment was more effective on hormone receptor-negative patients (HR 0.73; 95% CI: 0.58-0.93; p=0.010). In addition, the combination of PI3K/Akt/mTOR inhibitors with trastuzumab slightly increased the risk of some adverse events, such as neutropenia, leukopenia, fatigue, and anemia. CONCLUSIONS: The combination treatments of PI3K/Akt/mTOR inhibitors and trastuzumab for PI3K/Akt/mTOR inhibitors combined with trastuzumab treatments for patients with HER2-positive breast cancer can improve median progression-free survival while increasing the incidence of adverse events. It is still controversial based on the current evidence. Due to the limited number and quality of included studies, more high-quality studies are needed for further analysis. [ABSTRACT FROM AUTHOR]

Background: The emergence of de novo or intrinsic trastuzumab resistance is exceedingly high in breast cancer that is HER2 positive and correlates with an abundant cancer stem cell (CSC)-like population. We sought to examine the capacity of β-escin, an anti-inflammatory drug, to address trastuzumab resistance in HER2-positive breast cancer cells. Methods: The effect of β-escin on trastuzumab-resistant and -sensitive cell lines in vitro was evaluated for apoptosis, expression of HER2 family members, and impact on CSC-like properties. An in vivo model of trastuzumab-resistant JIMT-1 was used to examine the efficacy and toxicity of β-escin. Results: β-escin induced mitochondrial-mediated apoptosis accompanied by reactive oxygen species (ROS) production and increased active p18Bax fragmentation, leading to caspase-3/-7 activation. Attenuation of CSC-related features by β-escin challenge was accompanied by marked reductions in CD44high/CD24low stem-like cells and aldehyde dehydrogenase 1 (ALDH1) activity as well as hindrance of mammosphere formation. β-escin administration also significantly retarded tumor growth and angiogenesis in a trastuzumab-resistant JIMT-1 xenograft model via downregulation of CSC-associated markers and intracellular domain HER2. Importantly, β-escin selectively inhibited malignant cells and was less toxic to normal mammary cells, and no toxic effects were found in liver and kidney function in animals. Conclusions: Taken together, our findings highlight β-escin as a promising candidate for the treatment of trastuzumab-resistant HER2-positive breast cancers. [ABSTRACT FROM AUTHOR]

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer. [ABSTRACT FROM AUTHOR]

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The effectiveness of lapatinib (LAP) and trastuzumab (TRZ), the first-line therapies for HER2 + breast cancer, has been limited owing to the development of acquired resistance in patients with HER2 + . This study aimed to investigate the alterations in metabolic signatures in LAP-resistant HCC1954 and TRZ-resistant HCC1954 and pathways in human HER2 + breast cancer cells using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and enrichment analysis. The HCC1954 parental cells were sequentially treated 13 rounds with LAP or TRZ to develop resistant cells and then tested for their cytotoxicity using the MTT assay. Metabolites were prepared from HCC1954 parental (MBXWT), HCC1954-LAP (MBXLAP), and HCC1954-TRZ (MBXTRZ) cells prior to LC-HRMS, chemometric, enrichment, and joint pathway analyses. LAP- and TRZ-resistant cells were successfully developed from HCC1954, and 29 and 17 differentially expressed metabolites (DEMs) were identified between MBXWT-MBXLAP and MBXWT-MBXTRZ, respectively. The analysis of DEMs between MBXWT and MBXLAP revealed significant enrichment in D-amino acid metabolism, while MBXWT and MBXTRZ identified valine, leucine, isoleucine biosynthesis, ascorbate, and aldarate metabolism. Joint pathway enrichment analysis of LAP-resistant DEMs and differentially expressed genes (DEGs) showed enrichment in glutathione metabolism, while that of TRZ-resistance and DEGs showed enrichment in carbohydrate metabolism, namely pentose and glucuronate interconversions, starch and sucrose metabolism, and galactose metabolism. The findings from this study indicate considerable metabolic changes in LAP- and TRZ-resistant HCC1954 cells, which are crucial for understanding the resistance mechanisms and developing strategies to overcome these problems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: The aim of this study was to explore the role of galectin‐3 in human epidermal growth factor receptor 2 (HER2)‐positive breast cancer cells and the potential mechanism. Methods: Kaplan–Meier (KM)‐plot and The Cancer Genome Atlas (TCGA) databases were used to study the role of galectin‐3 in the prognosis of HER2‐positive breast cancer. The effects of galectin‐3 on cell proliferation, migration, invasion, and colony formation ability in HER2‐positive breast cancer cells were examined. The relationship between galectin‐3 and important components in the HER2 pathways, including HER2, epidermal growth factor receptor (EGFR), protein kinase B (AKT), and phosphatase and tensin homolog (PTEN), was further studied. Lentivirus and CRISPR/Cas9 were used to construct stable cell lines. Cell counting kit‐8 (CCK‐8) and apoptosis assays were used to study the relationship between galectin‐3 and trastuzumab. The effect of galectin‐3 on cell stemness was studied by mammosphere formation assay. The effects of galectin‐3 on stemness biomarkers and the Notch1 pathway were examined. Tumorigenic models were used to evaluate the effects of galectin‐3 on tumorigenesis and the therapeutic effect of trastuzumab in vivo. Results: HER2‐positive breast cancer patients with a high expression level of LGALS3 (the gene encoding galectin‐3) messenger RNA (mRNA) showed a poor prognosis. Galectin‐3 promoted cancer malignancy through phosphoinositide 3‐kinase (PI3K)/AKT signaling pathway activation and upregulated stemness by activating the Notch1 signaling pathway in HER2‐positive breast cancer cells. These two factors contributed to the enhancement of trastuzumab resistance in cells. Knockout of LGALS3 had a synergistic therapeutic effect with trastuzumab both in vitro and in vivo. Conclusions: Galectin‐3 may represent a prognostic predictor and therapeutic target for HER2‐positive breast cancer. [ABSTRACT FROM AUTHOR]

HER2‐positive breast cancer is an aggressive subtype of breast cancer, characterized by high malignancy and poor prognosis. Trastuzumab, the first HER2‐targeted monoclonal antibody therapy, has a crucial role in a curative setting in HER2‐positive breast cancer. However, frequent drug resistance inhibits its clinical efficacy. Herein, by performing circular RNA (circRNA) profiling, we identified a novel circRNA, circ‐BGN, as a key contributor in trastuzumab resistance. Circ‐BGN was evidently increased in trastuzumab‐resistant breast cancer cells and tissues, linking to poor overall survival. Knockdown of circ‐BGN inhibited breast cancer cell viability and notably restored its sensitivity to trastuzumab. Further, we found that circ‐BGN could directly bind to OTUB1 and SLC7A11, enhancing OTUB1‐mediated SLC7A11 deubiquitination and thereby inhibiting ferroptosis, a newly recognized form of cell death that is distinct from apoptosis, necrosis, and autophagy. Moreover, erastin, a small‐molecule ferroptosis inducer, could effectively restore the anti‐tumor effect of trastuzumab. Pre‐clinically, the orthotopic tumor model showed that erastin significantly reduced tumor volume generated by trastuzumab‐resistant breast cancer cells, which was more pronounced after combined circ‐BGN knockdown. Collectively, our data reveal a novel circRNA controlling trastuzumab resistance via regulation of ferroptosis, providing a promising therapeutic strategy for trastuzumab‐resistant breast cancer patients. [ABSTRACT FROM AUTHOR]

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

Trastuzumab resistance presents a significant challenge in the treatment of HER2+ breast cancer, necessitating the investigation of combination therapies to overcome this resistance. Honokiol, a compound with broad anticancer activity, has shown promise in this regard. This study aims to discover the effect of honokiol in increasing trastuzumab sensitivity in HER2+ trastuzumab-resistant breast cancer cells HCC1954 and the underline mechanisms behind. A bioinformatics study performed to explore the most potential target hub gene for honokiol in HER2+ breast cancer. Honokiol, trastuzumab and combined treatment cytotoxicity activity was then evaluated in both parental HCC1954 and trastuzumab resistance (TR-HCC1954) cells using MTT assay. The expression levels of these hub genes were then analyzed using qRT-PCR and those that could not be analyzed were subjected to molecular docking to determine their potential. Honokiol showed a potent cytotoxicity activity with an IC 50 of 41.05 μM and 69.61 μM in parental HCC1954 and TR-HCC1954 cell line respectively. Furthermore, the combination of honokiol and trastuzumab resulted in significant differences in cytotoxicity in TR-HCC1954 cells at specific concentrations. Molecular docking and the qRT-PCR showed that the potential ERα identified from the bioinformatics analysis was affected by the treatment. Our results show that honokiol has the potential to increase the sensitivity of trastuzumab in HER2+ trastuzumab resistant breast cancer cell line HCC1954 by affecting regulating estrogen receptor signaling. Further research is necessary to validate these findings. [Display omitted] • Using bioinformatics to explore the potential target hub gene of honokiol in HER2+ trastuzumab-resistant breast cancer cells. • Honokiol increased the sensitivity of trastuzumab in trastuzumab-resistant HCC1954 breast cancer cells. • ERα as the potential target of honokiol in overcoming breast cancer resistance towards trastuzumab. [ABSTRACT FROM AUTHOR]

The human epidermal growth factor receptor-2 (ERBB2; formerly HER2)isoform ERBB2ΔEx16 (ERBB2d16) was oncogenic by mediating epithelial-mesenchymal transition (EMT), immune evasion, and resistance cell death to the anti-HER2 (trastuzumab) therapy. However, its physiological implications in gastric cancer were unclear. In this study, we examined a total of 110 patients with either locally advanced or metastatic HER2+ gastric cancer for the expression of ERBB2d16 and EMT markers, and the infiltration of CD3+ T cells in tumor tissues, and evaluated their relevance with the responses to the standard chemotherapy plus trastuzumab according to the RECIST criteria. We found that the ERBB2d16 isoform was present at a relatively high level in about half of the tumor samples examined (53/110) and an elevated ERBB2d16/ERBB2 ratio was positively associated with the expression of high E-cadherin and low vimentin indicating EMT, and with poor CD3+ T cell infiltration and strong intratumoral expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) as well as reduced diversity of T cell receptor clones. Moreover, the progression-free survival and overall survival of patients treated with trastuzumab were substantially shorter in those with a high ERBB2d16/ERBB2 ratio. In agreement, analysis by Cox proportional hazards models confirmed that high ERBB2d16 expression was a risk factor associated with an adverse prognosis. Thus, our data fit well with an oncogenic role of ERBB2d16 in gastric cancer by promoting EMT and immunosuppression. We also found that ERBB2d16 expression resists gastric cell death in patients treated with trustuzumab, and the ERBB2d16/ERBB2 ratio may serve as a novel prognostic maker for patients with gastric cancer that receive trastuzumab therapy. [ABSTRACT FROM AUTHOR]

Background and purpose: As the targeted antibody of human epidermal growth factor receptor 2 (HER2), trastuzumab resistance gets more and more attention. This study aimed to explore the role of miR-375 in the occurrence of trastuzumab resistance in breast cancer cells through regulating epithelial-mesenchymal transition (EMT) by targeting Yes-associated protein 1 (YAP1). Methods: Trastuzumab-resistant breast cancer cell line was established. miR-375 mimic and recombinant plasmid YAP1 were transfected into SK-BR-3R cells. The sensitivities of each breast cancer cell line to trastuzumab were detected by MTT assay, and proliferation was detected by colony formation assay. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were applied to detect mRNA and protein expressions of miR-375, vimentin, E-cadherin and YAP1. Dual-luciferase reporter gene assay was conducted to verify the role of miR-375 in regulation of YAP1 transcription. A total of 25 breast cancer tissues from patients were collected to detect correlation in clinical performance. Results: Compared with NC group, after transfection with miR-375 mimic, the sensitivity to trastuzumab (P

The human epidermal growth factor receptor-2 (ERBB2; formerly HER2)isoform ERBB2ΔEx16 (ERBB2d16) was oncogenic by mediating epithelial-mesenchymal transition (EMT), immune evasion, and resistance cell death to the anti-HER2 (trastuzumab) therapy. However, its physiological implications in gastric cancer were unclear. In this study, we examined a total of 110 patients with either locally advanced or metastatic HER2+ gastric cancer for the expression of ERBB2d16 and EMT markers, and the infiltration of CD3+ T cells in tumor tissues, and evaluated their relevance with the responses to the standard chemotherapy plus trastuzumab according to the RECIST criteria. We found that the ERBB2d16 isoform was present at a relatively high level in about half of the tumor samples examined (53/110) and an elevated ERBB2d16/ERBB2 ratio was positively associated with the expression of high E-cadherin and low vimentin indicating EMT, and with poor CD3+ T cell infiltration and strong intratumoral expression of programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) as well as reduced diversity of T cell receptor clones. Moreover, the progression-free survival and overall survival of patients treated with trastuzumab were substantially shorter in those with a high ERBB2d16/ERBB2 ratio. In agreement, analysis by Cox proportional hazards models confirmed that high ERBB2d16 expression was a risk factor associated with an adverse prognosis. Thus, our data fit well with an oncogenic role of ERBB2d16 in gastric cancer by promoting EMT and immunosuppression. We also found that ERBB2d16 expression resists gastric cell death in patients treated with trustuzumab, and the ERBB2d16/ERBB2 ratio may serve as a novel prognostic maker for patients with gastric cancer that receive trastuzumab therapy.

The specific human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibody trastuzumab shows considerable clinical efficacy in patients with HER2-overexpressing breast cancer. However, about 20% of patients who receive trastuzumab in the adjuvant setting relapse, and approximately half of patients with metastatic HER2-positive breast cancer develop resistance to trastuzumab within 1 year. Although the mechanism of trastuzumab resistance has been explored broadly, whether and how angiogenesis participates in trastuzumab resistance is unclear. Here, we examined the association between angiogenesis and trastuzumab resistance by using a trastuzumab-resistant cell line (SKBR3-TR). Compared with that from the parental trastuzumab-sensitive SKBR3 cells, the culture supernatant from SKBR3-TR cells significantly increased the sprouting of endothelial cells. To identify intercellular features that contribute to the induction of endothelial tube formation, proteomics revealed that α-crystallin B chain (αB-crystallin) was upregulated in SKBR3-TR cells. Moreover, silencing of αB-crystallin significantly repressed SKBR3-TR-induced tube formation, and knockdown of αB-crystallin in SKBR3-TR cells suppressed the activation of mechanistic target of rapamycin (mTOR) in endothelial cells. In addition, treatment with rapamycin, an inhibitor of mTOR, reversed the SKBR3-TR-induced promotion of tube formation. In summary, αB-crystallin enhanced the ability of SKBR3-TR cells to activate mTOR in endothelial cells and thus promote angiogenesis. • Increased αB-crystallin level of SKBR3-TR cells promoted tube formation by HAECs. • Silencing αB-crystallin in SKBR3-TR cells suppresses p-mTOR in endothelial cells. • Rapamycin suppressed tube formation in HAECs induced by SKBR3-TR cells. [ABSTRACT FROM AUTHOR]

HER2 amplification/overexpression is a common driver in a variety of cancers including gallbladder cancer (GBC). For patients with metastatic GBC, chemotherapy remains the standard of care with limited efficacy. The combination of HER2 antibody trastuzumab plus chemotherapy is the frontline treatment option for patients with HER2-positive breast cancer and gastric cancer. Recently, this regime also showed antitumor activity in HER2-positive GBC. However, resistance to this regime represents a clinical challenge. Camrelizumab is a novel PD-1 antibody approved for Hodgkin lymphoma and hepatocellular carcinoma in China. In this study, we presented a HER2-positive metastatic GBC patient who was refractory to trastuzumab plus chemotherapy but experienced significant clinical benefit after the addition of camrelizumab. Our case highlights the potential of immunotherapy in combination with HER2-targeted therapy in HER2-positive GBC. We also demonstrated that two immune-related adverse events (irAEs) associated with camrelizumab can be managed with an anti-VEGF agent apatinib. This case not only highlights the importance of irAE management in patients treated with camrelizumab, but also demonstrates the potential of PD-1 antibody plus trastuzumab in HER2-positive GBC patients who have developed resistance to chemotherapy and trastuzumab-based targeted therapy. [ABSTRACT FROM AUTHOR]

Abstract Trastuzumab resistance in HER2-positive breast cancer is associated with a poorer prognosis. HSP90 is thought to play a major role in such resistance, but N-terminal inhibitors of this target have had little success. We sought to investigate the utility of NCT-547, a novel, rationally-designed C-terminal HSP90 inhibitor in the context of overcoming trastuzumab resistance. NCT-547 treatment significantly induced apoptosis without triggering the heat shock response (HSR), accompanied by caspase-3/− 7 activation in both trastuzumab-sensitive and -resistant cells. NCT-547 effectively promoted the degradation of full-length HER2 and truncated p95HER2, while also attenuating hetero-dimerization of HER2 family members. The impairment of cancer stem-like traits was observed with reductions in ALDH1 activity, the CD24low/CD44high subpopulation, and mammosphere formation in vitro and in vivo. NCT-547 was an effective inhibitor of tumor growth and angiogenesis, and no toxic outcomes were found in initial hepatic and renal analysis. Our findings suggest that NCT-547 may have applications in addressing trastuzumab resistance in HER2-positive breast cancer.

Abstract Background Although trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Recent evidence suggests that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and chemoresistance. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is not well established to date. In this research, we identified the differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer. Methods LncRNA microarray and qRT-PCR were performed to identify the dysregulated lncRNAs. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of exosomal AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1). Bioinformatics prediction, RNA fluorescence in situ hybridization (RNA-FISH) and immunoprecipitation assays were performed to identify the direct interactions between AFAP1-AS1 and other associated targets, such as AU-binding factor 1 (AUF1) and ERBB2. Finally, a series gain- or loss-functional assays were done to prove the precise role of AFAP1-AS1 in trastuzumab resistance. Results AFAP1-AS1 was screened out due to its higher expression in trastuzumab-resistant cells compared to sensitive cells. Increased expression of AFAP1-AS1was associate with poorer response and shorter survival time of breast cancer patients. AFAP1-AS1 was upregulated by H3K27ac modification at promoter region, and knockdown of AFAP1-AS1 reversed trastuzumab resistance. Moreover, extracellular AFAP1-AS1 secreted from trastuzumab resistant cells was packaged into exosomes and then disseminated trastuzumab resistance of receipt cells. Mechanically, AFAP1-AS1 was associated with AUF1 protein, which further promoted the translation of ERBB2 without influencing the mRNA level. Conclusion Exosomal AFAP1-AS1 could induce trastuzumab resistance through associating with AUF1 and promoting ERBB2 translation. Therefore, AFAP1-AS1 level may be useful for prediction of trastuzumab resistance and breast cancer treatment.

HER2 amplification/overexpression is a common driver in a variety of cancers including gallbladder cancer (GBC). For patients with metastatic GBC, chemotherapy remains the standard of care with limited efficacy. The combination of HER2 antibody trastuzumab plus chemotherapy is the frontline treatment option for patients with HER2-positive breast cancer and gastric cancer. Recently, this regime also showed antitumor activity in HER2-positive GBC. However, resistance to this regime represents a clinical challenge. Camrelizumab is a novel PD-1 antibody approved for Hodgkin lymphoma and hepatocellular carcinoma in China. In this study, we presented a HER2-positive metastatic GBC patient who was refractory to trastuzumab plus chemotherapy but experienced significant clinical benefit after the addition of camrelizumab. Our case highlights the potential of immunotherapy in combination with HER2-targeted therapy in HER2-positive GBC. We also demonstrated that two immune-related adverse events (irAEs) associated with camrelizumab can be managed with an anti-VEGF agent apatinib. This case not only highlights the importance of irAE management in patients treated with camrelizumab, but also demonstrates the potential of PD-1 antibody plus trastuzumab in HER2-positive GBC patients who have developed resistance to chemotherapy and trastuzumab-based targeted therapy.

Platelet-derived growth factor receptor (PDGFR) plays an essential role in the proliferation and invasion of malignant cancer cells. However, the functional role of PDGFR alpha (PDGFRA) in HER2-positive (HER2+) breast cancer has not been fully clarified yet. Thus, the objective of this study was to investigate the clinical significance of PDGFRA and the therapeutic potential of PDGFR inhibitors as part of an effort to overcome trastuzumab (TRZ) resistance. Aberrant PDGFRA expression is closely associated with decreased survival in HER2+ breast cancers. Therefore, we established BT474 trastuzumab-sensitive (TRZ_S) and trastuzumab-resistant (TRZ_R) cells to investigate the association between PDGFR signaling and TRZ resistance. We found that PDGFRA was significantly upregulated in the BT474 TRZ_R cells. In addition, IL-6 expression, which was also found to be upregulated in the TRZ_R cells, was induced by PDGFC, a ligand of PDGFR. Next, we investigated the effects of ponatinib and sunitinib, PDGFR inhibitors, on the BT474 TRZ_R and HCC1954 (TRZ-resistant cell line) cells. These inhibitors decreased cell viability and migration in a dose-dependent manner. Additionally, IL-6 expression was decreased by ponatinib in both the BT474 TRZ_R and HCC1954 cells. In contrast, IL-6 was not suppressed by TRZ, implying that the PDGFRA/STAT3/IL-6 axis is associated with resistance to TRZ. In addition, we found that STAT3 and ERK phosphorylation were increased in the BT474 TRZ_R cells. IL-6 expression was suppressed by a STAT3 inhibitor, indicating that IL-6 expression is modulated downstream of STAT3. Taken together, these results suggest that PDGFRA could serve as a therapeutic target to overcome TRZ resistance.

Background: Trastuzumab resistance is a serious clinical problem in the treatment of HER2-positive breast cancer (BC). The lncRNA ZNF649-AS1 was previously found to promote HER2-positive BC trastuzumab resistance. The study aims to explore the molecular mechanism of ZNF649-AS1 in HER2-positive BC trastuzumab resistance., Methods: Tumor tissue and peripheral blood samples were collected from 20 HER2-positive BC patients with trastuzumab-resistant and non-resistant, respectively. Trastuzumab-resistant BC cell lines SKBR-3-TR and BT474-TR were established. RIP was employed to confirm the binding of ZNF649-AS1, PRPF8 and exocyst complex component 7 (EXOC7). RNA expression of EXOC7-L (Full length of EXOC7) and EXOC7-S (Spliceosome of EXOC7) were detected using agarose gel electrophoresis. Expressions of macrophage markers CD68 + CD206 + were measured by flow cytometry., Results: ZNF649-AS1 expression was upregulated in HER2-positive BC trastuzumab resistance. ZNF649-AS1 downregulation inhibited trastuzumab resistance in HER2-positive BC. ZNF649-AS1 regulated EXOC7 alternative splicing by binding with PRPF8. EXOC7-S knockdown suppressed trastuzumab resistance and TAM-dependent PD-L1 expression in HER2-positive BC. EXOC7-S overexpression abolished the effects of ZNF649-AS1 knockdown on trastuzumab resistance and TAM-dependent PD-L1 expression in HER2-positive BC., Conclusion: ZNF649-AS1 promoted trastuzumab resistance and TAM-dependent PD-L1 expression in HER2-positive BC via promoting alternative splicing of EXOC7 by PRPF8., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Approximately 50-74% of patients with metastatic HER2-positive breast cancer do not respond to trastuzumab, with 75% of treated patients experiencing disease progression within a year. The combination of pyrotinib and capecitabine has showed efficacy in these patients. This study evaluates the efficacy and safety of pyrotinib combined with metronomic vinorelbine for trastuzumab-pretreated HER2-positive advanced breast cancer patients., Materials and Methods: In this phase 2 trial, patients aged 18-75 years with HER2-positive advanced breast cancer who had previously failed trastuzumab treatment were enrolled to receive pyrotinib 400mg daily in combination with vinorelbine 40mg thrice weekly. The primary endpoint was progression-free survival (PFS), while secondary endpoints included objective response rate (ORR), disease control rate (DCR), overall survival (OS), and safety., Results: From October 21, 2019, to January 21, 2022, 36 patients were enrolled and received at least one dose of study treatment. At the cut-off date, 20 experienced disease progression or death. With a median follow-up duration of 35 months, the median PFS was 13.5 months (95% CI: 8.3-18.5). With all patients evaluated, an ORR of 38.9% (95% CI: 23.1-56.5%) and a DCR of 83.3% (95% CI: 67.2-93.6%) were achieved. The median OS was not reached. Grade 3 adverse events (AEs) were observed in 17 patients, with diarrhea being the most common (27.8%), followed by vomiting (8.3%) and stomachache (5.6%). There were no grade 4/5 AEs., Conclusion: Pyrotinib combined with metronomic vinorelbine showed promising efficacy and an acceptable safety profile in HER2-positive advanced breast cancer patients after trastuzumab failure.