Proteomic Characterization of a 3D HER2+ Breast Cancer Model Reveals the Role of Mitochondrial Complex I in Acquired Resistance to Trastuzumab.

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.

Genomic Alterations Correlated to Trastuzumab Resistance and Clinical Outcomes in HER2+/HR- Breast Cancers of Patients Living in Northwestern China.

Anti-HER2 therapy has significantly improved the survival rates of patients with HER2+ breast cancer. However, a subset of these patients eventually experience treatment failure, and the underlying genetic mechanisms remain largely unexplored. This underscores the need to investigate the genomic heterogeneity of HER2+ breast cancer. In this study, we focus on HER2+/HR- breast cancer, as it differs from HER2+/HR+ breast cancer in terms of genetic and biological characteristics. We performed gene-targeted genome sequencing on 45 HER2+/HR- breast cancer samples and identified 650 mutations across 268 cancer-related genes. TP53 (71.1%) and PIK3CA (35.6%) were the most frequently mutated genes in our sample. Additionally, ERBB2 (77.8%), CDK12 (42.2%), and MYC (11.1%) exhibited a high frequency of copy number amplifications (CNAs). Comparative analysis with two other HER2+/HR- breast cancer cohorts revealed that our cohort had higher genetic variation rates in ARID1A, PKHD1, PTPN13, FANCA, SETD2, BRCA2, BLM, STAG2, FAT1, TOP2A, POLE, ATM, KMT2B, FGFR4, and EPAS1. Notably, in our cohort, NF1 and ATM mutations were more prevalent in trastuzumab-resistant patients (NF1, p=0.016; ATM, p=0.006) and were associated with primary trastuzumab resistance (NF1, p=0.042; ATM, p=0.021). Moreover, patients with NF1 mutations (p=0.009) and high histological grades (p=0.028) were more likely to experience early relapse. Ultimately, we identified a unique cancer-related gene mutation profile and a subset of genes associated with primary resistance to trastuzumab and RFS in patients with HER2+/HR- breast cancer in Northwest China. These findings could lay the groundwork for future studies aimed at elucidating the mechanisms of resistance to trastuzumab and improving HER2-targeted treatment strategies.

Integrin ß3 Reprogramming Stemness in HER2-Positive Breast Cancer Cell Lines.

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 ITGß3 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 ITGß3 enhances the expression of these stem cell markers, while silencing ITGß3 reduces their expression, suggesting a major role for ITGß3 in maintaining stemness and resistance. Further analysis reveals that ITGß3 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 ITGß3 in mediating stemness and trastuzumab resistance through Notch signalling in HER2-positive breast cancer, offering new approaches for enhancing treatment efficacy.

Nanocage-incorporated engineered destabilized 3'UTR ARE of ERBB2 inhibits tumor growth and liver and lung metastasis in EGFR T790M osimertinib- and trastuzumab-resistant and ERBB2-expressing NSCLC via the reduction of ERBB2.

Non-small cell lung cancer (NSCLC) caused more deaths in 2017 than breast cancer, prostate, and brain cancers combined. This is primarily due to their aggressive metastatic nature, leading to more fatal rates of cancer patients. Despite this condition, there are no clinically approved drugs that can target metastasis. The NSCLC with EGFR T790M-overexpressing HER2 shows the resistance to osimertinib and trastuzumab starting 10-18 months after the therapy, and thus prospects are grim to these patients. To target the recalcitrant ERBB2 driver oncogene, we developed two engineered destabilizing 3'UTR ERBB2 constructs that degrade the endogenous ERBB2 transcript and proteins by overwriting the encoded endogenous ERBB2 mRNA with the destabilizing message. When iron oxide nanocages (IO nanocages) were used as vehicles to deliver them to tumors and whole tissues in mice bearing tumors, it was well tolerated and safe and caused no genome rearrangement whereas they were integrated into genome deserts (non-coding regions). We achieved significant reduction of the primary tumor volume with desARE3'UTRERBB2-30, achieving 50% complete tumor lysis and inhibiting 60%-80% of liver metastasis, hepatomegaly, and 90% of lung metastasis, through ERBB2 downregulation. These constructs were distributed robustly into tumors, livers, lungs, kidneys, and spleen and mildly in the brain and not in the heart. They caused no abnormality in both short- and long-term administrations as well as in healthy mice. In summary, we accomplished significant breakthrough for the therapeutics of intractable lung cancer patients whose cancers become resistant and metastasize.

M6A RNA Methylation-Mediated Dysregulation of AGAP2-AS1 Promotes Trastuzumab Resistance of Breast Cancer.

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.

Bioinformatics and In Vitro Study Reveal ERα as The Potential Target Gene of Honokiol to Enhance Trastuzumab Sensitivity in HER2+ Trastuzumab-Resistant Breast Cancer Cells.

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 IC50 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.

Combined transcriptome and proteome analysis reveals MSN and ARFIP2 as biomarkers for trastuzumab resistance of breast cancer.

PURPOSE: HER2-positive breast cancer (BC) accounts for 20-30% of all BC subtypes and is linked to poor prognosis. Trastuzumab (Tz), a humanized anti-HER2 monoclonal antibody, is a first-line treatment for HER2-positive breast cancer which faces resistance challenges. This study aimed to identify the biomarkers driving trastuzumab resistance. METHODS: Differential expression analysis of genes and proteins between trastuzumab-sensitive (TS) and trastuzumab-resistant (TR) cells was conducted using RNA-seq and iTRAQ. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were used to study their functions. The prognostic significance and protein levels of ARFIP2 and MSN were evaluated using online tools and immunohistochemistry. Sensitivity of MSN and ARFIP2 to other therapies was assessed using public pharmacogenomics databases and the R language. RESULTS: Five genes were up-regulated, and nine genes were down-regulated in TR cells at both transcriptional and protein levels. Low ARFIP2 and high MSN expression linked to poor BC prognosis. MSN increased and ARFIP2 decreased in TR patients, correlating with shorter OS. MSN negatively impacted fulvestrant and immunotherapy sensitivity, while ARFIP2 had a positive impact. CONCLUSION: Our findings suggest that MSN and ARFIP2 could serve as promising biomarkers for predicting response to Tz, offering valuable insights for future research in the identification of diagnostic and therapeutic targets for BC patients with Tz resistance.

BCAR4 facilitates trastuzumab resistance and EMT in breast cancer via sponging miR-665 and interacting with YAP1.

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-ß 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.

The HSP90 inhibitor HVH-2930 exhibits potent efficacy against trastuzumab-resistant HER2-positive breast cancer.

Rationale: Resistance to targeted therapies like trastuzumab remains a critical challenge for HER2-positive breast cancer patients. Despite the progress of several N-terminal HSP90 inhibitors in clinical trials, none have achieved approval for clinical use, primarily due to issues such as induction of the heat shock response (HSR), off-target effects, and unfavorable toxicity profiles. We sought to examine the effects of HVH-2930, a novel C-terminal HSP90 inhibitor, in overcoming trastuzumab resistance. Methods: The effect of HVH-2930 on trastuzumab-sensitive and -resistant cell lines in vitro was evaluated in terms of cell viability, expression of HSP90 client proteins, and impact on cancer stem cells. An in vivo model with trastuzumab-resistant JIMT-1 cells was used to examine the efficacy and toxicity of HVH-2930. Results: HVH-2930 was rationally designed to fit into the ATP-binding pocket interface cavity of the hHSP90 homodimer in the C-terminal domain of HSP90, stabilizing its open conformation and hindering ATP binding. HVH-2930 induces apoptosis without inducing the HSR but by specifically suppressing the HER2 signaling pathway. This occurs with the downregulation of HER2/p95HER2 and disruption of HER2 family member heterodimerization. Attenuation of cancer stem cell (CSC)-like properties was associated with the downregulation of stemness factors such as ALDH1, CD44, Nanog and Oct4. Furthermore, HVH-2930 administration inhibited angiogenesis and tumor growth in trastuzumab-resistant xenograft mice. A synergistic effect was observed when combining HVH-2930 and paclitaxel in JIMT-1 xenografts. Conclusion: Our findings highlight the potent efficacy of HVH-2930 in overcoming trastuzumab resistance in HER2-positive breast cancer. Further investigation is warranted to fully establish its therapeutic potential.

An Imbalance in Histone Modifiers Induces tRNA-Cys-GCA Overexpression and tRF-27 Accumulation by Attenuating Promoter H3K27me3 in Primary Trastuzumab-Resistant Breast Cancer.

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.

Unveiling Alterations of Epigenetic Modifications and Chromatin Architecture Leading to Lipid Metabolic Reprogramming during the Evolutionary Trastuzumab Adaptation of HER2-Positive Breast Cancer.

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies.

Peptidylprolyl isomerase D circular RNA sensitizes breast cancer to trastuzumab through remodeling HER2 N4-acetylcytidine modification.

Human epidermal growth factor receptor 2 (HER2) overexpression and activation are crucial for trastuzumab resistance in HER2-positive breast cancer; however, the potential regulatory mechanism of HER2 is still largely undetermined. In this study, a novel circular RNA derived from peptidylprolyl isomerase D (PPID) is identified as a negative regulator of trastuzumab resistance. Circ-PPID is highly stable and significantly downregulated in trastuzumab-resistant cells and tissues. Restoration of circ-PPID markedly enhances HER2-positive breast cell sensitivity to trastuzumab in vitro and in vivo. Circ-PPID directly binds to N-acetyltransferase 10 (NAT10) in the nucleus and blocks the interaction between NAT10 and HER2 mRNA, reducing N4-acetylcytidine (ac4C) modification on HER2 exon 25, leading to HER2 mRNA decay. Intriguingly, the subcellular localization of circ-PPID differs between trastuzumab-sensitive and -resistant cells. Circ-PPID in trastuzumab-resistant cells is located more in the cytoplasm, mainly due to the upregulation of Exportin 4 (XPO4), which results in the loss of spatial conditions for circ-PPID to bind to nuclear NAT10. Taken together, our data suggest that circ-PPID is a previously unappreciated ac4C-dependent HER2 epigenetic regulator, providing a promising therapeutic direction for overcoming trastuzumab resistance in clinical setting.

Molecular Atlas of HER2+ Breast Cancer Cells Treated with Endogenous Ligands: Temporal Insights into Mechanisms of Trastuzumab Resistance.

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.

Tyrosine phosphorylation-mediated YAP1-TFAP2A interactions coordinate transcription and trastuzumab resistance in HER2+ breast cancer.

Trastuzumab resistance in HER2+ breast cancer (BC) is the major reason leading to poor prognosis of BC patients. Oncogenic gene overexpression or aberrant activation of tyrosine kinase SRC is identified to be the key modulator of trastuzumab response. However, the detailed regulatory mechanisms underlying SRC activation-associated trastuzumab resistance remain poorly understood. In the present study, we discover that SRC-mediated YAP1 tyrosine phosphorylation facilitates its interaction with transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha, TFAP2A), which in turn promotes YAP1/TEAD-TFAP2A (YTT) complex-associated transcriptional outputs, thereby conferring trastuzumab resistance in HER2+ BC. Inhibition of SRC kinase activity or disruption of YTT complex sensitizes cells to trastuzumab treatment in vitro and in vivo. Additionally, we also identify YTT complex co-occupies the regulatory regions of a series of genes related to trastuzumab resistance and directly regulates their transcriptions, including EGFR, HER2, H19 and CTGF. Moreover, YTT-mediated transcriptional regulation is coordinated by SRC kinase activity. Taken together, our study reveals that SRC-mediated YTT complex formation and transcriptions are responsible for multiple mechanisms associated with trastuzumab resistance. Therefore, targeting HER2 signaling in combination with the inhibition of YTT-associated transcriptional outputs could serve as the treatment strategy to overcome trastuzumab resistance caused by SRC activation.

Liposome-enabled bufalin and doxorubicin combination therapy for trastuzumab-resistant breast cancer with a focus on cancer stem cells.

Breast cancer stem cells (BCSCs) play a key role in therapeutic resistance in breast cancer treatments and disease recurrence. This study aimed to develop a combination therapy loaded with pH-sensitive liposomes to kill both BCSCs and the okbulk cancer cells using trastuzumab-sensitive and resistant human epidermal growth factor receptor 2 positive (HER2+) breast cancer cell models. The anti-BCSCs effect and cytotoxicity of all-trans retinoic acid, salinomycin, and bufalin alone or in combination with doxorubicin were compared in HER2+ cell line BT-474 and a validated trastuzumab-resistant cell line, BT-474R. The most potent anti-BCSC agent was selected and loaded into a pH-sensitive liposome system. The effects of the liposomal combination on BCSCs and bulk cancer cells were assessed. Compared with BT-474, the aldehyde dehydrogenase positive BCSC population was elevated in BT-474R (3.9 vs. 23.1%). Bufalin was the most potent agent and suppressed tumorigenesis of BCSCs by ∼50%, and showed strong synergism with doxorubicin in both BT-474 and BT-474R cell lines. The liposomal combination of bufalin and doxorubicin significantly reduced the BCSC population size by 85%, and inhibited both tumorigenesis and self-renewal, although it had little effect on the migration and invasiveness. The cytotoxicity against the bulk cancer cells was also enhanced by the liposomal combination than either formulation alone in both cell lines (p < 0.001). The liposomal bufalin and doxorubicin combination therapy may effectively target both BCSCs and bulk cancer cells for a better outcome in trastuzumab-resistant HER2+ breast cancer.

Identification of a novel potent CDK inhibitor degrading cyclinK with a superb activity to reverse trastuzumab-resistance in HER2-positive breast cancer in vivo.

CDK12 is overexpressed in HER2-positive breast cancers and promotes tumorigenesis and trastuzumab resistance. Thus CDK12 is a good therapeutic target for the HER2-positive breast tumors resistant to trastuzumab. We previously reported a novel purine-based CDK inhibitor with an ability to degrade cyclinK. Herein, we further explored and synthesized new derivatives, and identified a new potent pan-CDK inhibitor degrading cyclinK (32e). Compound 32e potently inhibited CDK12/cyclinK with IC50 = 3 nM, and suppressed the growth of the both trastuzumab-sensitive and trastuzumab-resistant HER2-positive breast cancer cell lines (GI50's = 9-21 nM), which is superior to a potent, clinical pan-CDK inhibitor dinaciclib. Moreover, 32e (10, 20 mg/kg, ip, twice a week) showed a dose-dependent inhibition of tumor growth and a more dramatic anti-cancer effect than dinaciclib in mouse in vivo orthotopic breast cancer model of trastuzumab-resistant HCC1954 cells. Kinome-wide inhibition profiling revealed that 32e at 1 µM exhibits a decent selectivity toward CDK-family kinases including CDK12 over other wildtype protein kinases. Quantitative global proteomic analysis of 32e-treated HCC1954 cells demonstrated that 32e also showed a decent selectivity in degrading cyclinK over other cyclins. Compound 32e could be developed as a drug for intractable trastuzumab-resistant HER2-positive breast cancers. Our current study would provide a useful insight in designing potent cyclinK degraders.

Thioridazine reverses trastuzumab resistance in gastric cancer by inhibiting S-phase kinase associated protein 2-mediated aerobic glycolysis.

BACKGROUND: Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2 (HER-2)-positive gastric cancer (GC). However, the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance. While S-phase kinase associated protein 2 (Skp2) overexpression has been implicated in the malignant progression of GC, its role in regulating trastuzumab resistance in this context remains uncertain. Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products, there has been a lack of successful commercialization of drugs specifically targeting Skp2. AIM: To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment. METHODS: Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells. Q-PCR, western blot, and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression. A cell counting kit-8 assay, flow cytometry, a amplex red glucose/glucose oxidase assay kit, and a lactate assay kit were utilized to measure the proliferation, apoptosis, and glycolytic activity of GC cells in vitro. A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo. RESULTS: The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab. Thioridazine demonstrated the ability to directly bind to Skp2, resulting in a reduction in Skp2 expression at both the transcriptional and translational levels. Moreover, thioridazine effectively inhibited cell proliferation, exhibited antiapoptotic properties, and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways. The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo, surpassing the efficacy of either monotherapy. CONCLUSION: Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance, particularly when used in conjunction with lapatinib. This compound has potential benefits for patients with Skp2-proficient tumors.

Albumin-bilirubin score predicts trastuzumab resistance in HER2-positive breast cancer.

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.

A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells.

Human epithelial growth factor receptor-2 (HER2) plays an oncogenic role in numerous tumors, including breast, gastric, and various other solid tumors. While anti-HER2 therapies are approved for the treatment of HER2-positive tumors, a necessity persists for creating novel HER2-targeted agents to resolve therapeutic resistance. Utilizing a synthetic nanobody library and affinity maturation, our study identified four anti-HER2 nanobodies that exhibited high affinity and specificity. These nanobodies recognized three distinct epitopes of HER2-ECD. Additionally, we constructed VHH-Fc and discovered that they facilitated superior internalization and showed moderate growth inhibition. Compared to the combination of trastuzumab and pertuzumab, the VHH-Fc combos or their combination with trastuzumab demonstrated greater or comparable antitumor activity in both ligand-independent and ligand-driven tumors. Most remarkably, A9B5-Fc, which targeted domain I of HER2-ECD, displayed significantly enhanced trastuzumab-synergistic antitumor efficacy compared to pertuzumab under trastuzumab-resistant conditions. Our findings offer anti-HER2 nanobodies with high affinity and non-overlapping epitope recognition. The novel nanobody-based HER2-targeted antibody, A9B5-Fc, binding to HER2-ECD I, mediates promising receptor internalization. It possesses the potential to serve as a potent synergistic partner with trastuzumab, contributing to overcoming acquired resistance.

The novel ß-TrCP protein isoform hidden in circular RNA confers trastuzumab resistance in HER2-positive breast cancer.

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-ß-TrCP, derived from the back-splicing of ß-TrCP exon 7 and 13, confers trastuzumab resistance by regulating NRF2-mediated antioxidant pathway in a KEAP1-independent manner. Concretely, circ-ß-TrCP encodes a novel truncated 343-amino acid peptide located in the nucleus, referred as ß-TrCP-343aa, which competitively binds to NRF2, blocks SCFß-TrCP-mediated NRF2 proteasomal degradation, and this protective effect of ß-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-ß-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 ß-TrCP-343aa and NRF2, expediting trastuzumab resistance. Collectively, our data demonstrate that circ-ß-TrCP-encoded ß-TrCP protein isoform drives HER2-targeted therapy resistance in a NRF2-dependent manner, which provides potential therapeutic targets for overcoming trastuzumab resistance.