Trastuzumab Resistance Research Articles

Sensitivity of HER2-amplified colorectal organotypic cancer spheroids at ex vivo resistance to panitumumab and trastuzumab.

68 Background: HER2 amplification is an emerging biomarker in colorectal cancer (CRC) with increased copy number associated with improved clinical outcomes to HER2 targeting. RAS/RAF wildtype CRC also benefit from use of epidermal growth factor receptor inhibition (EGFRi). The sequencing of EGFRi versus HER2 inhibition in low copy number HER2 amplified CRC remains uncertain. Patient-derived cancer organoids (PDCOs) allow an ex vivo method to assess treatment sensitivity. We examined treatment sensitivity of a HER2 amplified PDCO at baseline and following resistance to panitumumab and trastuzumab. Methods: Following IRB-approval, fresh CRC tissue was cultured to maturation. After expansion, subcultures were treated with stepwise (20%) increase to physiologic Cmax of panitumumab (230ug/mL) and trastuzumab (180ug/mL). Threshold for escalation was median relative growth of +20% at 96h. Sensitivity was assessed on primary culture (RC1), panitumumab resistance (RC1-P) and trastuzumab resistance (RC1-T) using 96h of physiologic Cmax panitumumab, trastuzumab, and combination trastuzumab/pertuzumab. Individual sphere response was assessed for change in mean NADH autofluorescence intensity and ratio of NADH/FAD signal. Response was assessed at 96h in comparison to control using effect size of Glass’s Delta (GΔ). Results: Molecular profiling revealed HER2 copy number of 14 with no concurrent alterations in RAS, RAF, or PIK3CA. Time to resistance was similar between panitumumab (55 days) and trastuzumab (51 days). RC1 had baseline growth (+116%) which was reduced with single agent panitumumab (+17%, GΔ=1.40) with intermediate sensitivity to trastuzumab (+48%, GΔ=0.95) and trastuzumab/pertuzumab (46%, GΔ=0.99). Normalized NADH/FAD ratio revealed significant metabolic response to panitumumab (-20%, GΔ=0.66) and trastuzumab/pertuzumab (-35%, GΔ=1.16) with insignificant effect of single agent trastuzumab (-14%, GΔ=0.46). Following resistance to panitumumab, RC1-P had persistent growth with trastuzumab (+68%) which improved in combination trastuzumab/pertuzumab (+34%, GΔ=1.16). Following resistance to trastuzumab, RC1-T was insensitive to EGFRi with panitumumab including persistent growth (+58%, GΔ=0.70) and unchanged metabolism (+2%, GΔ=-0.10). Conclusions: Therapeutic dose escalation in a single PDCO of HER2 amplified CRC suggests improved sensitivity to EGFRi and dual HER2 targeting with trastuzumab/pertuzumab. Resistance to EGFRi resulted in persistent sensitivity to dual HER2 inhibition using trastuzumab/pertuzumab, however resistance to single agent trastuzumab. Resistance to trastuzumab resulted in future insensitivity to EGFRi. Molecular profiling at resistance revealed no pathologic alterations in EGFR or ERBB2 signaling, with ongoing analysis of transcriptional changes by RNAseq.

Response to trastuzumab and investigation of expression profiles of matrix metalloproteinase-related proteins in primary breast cancer stem cells.

Breast cancer (BC) is the leading cause of cancer deaths in women. One of the reasons for the failure of BC treatment is reportedly the ineffectiveness of chemotherapeutic drugs against breast cancer stem-like cells (BCSCs). HER2 receptors have an important role in the self-renewal of BCSCs. Matrix metalloproteinase (MMP) and cytokine levels were found to be higher in BCSCs, which demonstrates their potential metastatic capacity. Therefore, the aim of this study was to evaluate the response of BCSCs to trastuzumab and to investigate the MMP levels in primary breast cancer cells and HER2+ BCSCs. Tumour tissue samples were obtained during surgical intervention from ten breast cancer patients, and primary culture cells were established from these tissues. Four major molecular subgroups were sorted from the primary culture: HER2+ BCSCs (CD44+CD24-HER2+), HER2- BCSCs (CD44+CD24-HER2-), HER2- primary culture cells (CD44+CD24+HER2-) and triple positive primary culture cells (CD44+CD24+HER2+). These cells were cultured and treated with trastuzumab, paclitaxel, carboplatin, and the combination of those three drugs for 96h. Cellular responses to these drugs were determined by XTT cytotoxicity test. MMPs and cytokine array analysis showed that MMPs and TIMP-1, TIMP-2 proteins were expressed more in HER2+ BCSCs than in primary culture. HER2- BCSCs were more resistant to drugs than HER2+ BCSCs. Our findings suggest that the presence of HER2- BCSCs may be responsible for primary trastuzumab resistance in HER2+ BC cell population. Further studies investigating the function of MMPs are needed for drug targeting of BCSCs.

NDUFA4L2 promotes trastuzumab resistance in HER2-positive breast cancer

Background:Trastuzumab (Herceptin) is the key systemic therapy for HER2-positive breast cancer. However, the initial response rate is limited to approximately 50% in patients. Moreover, most patients, especially at an advanced stage, eventually develop acquired resistance. Understanding the mechanisms of trastuzumab resistance is crucial for achieving better treatment outcome in this group of patients.Methods:A trastuzumab-resistant (TR) cell line was developed using the BT474 HER2-positive breast cancer cell line. Whole-transcriptome expression array was performed and the TR-related gene NDUFA4L2 was identified by differential expression analysis between BT474 and BT474-TR. Mitochondrial localization of NDUFA4L2 was confirmed by immunofluorescence and western blotting using mitochondrial fractionation. Mitochondrial function and energy metabolism were evaluated using Seahorse, ATP production, and lactate production assays, and cellular reactive oxygen species (ROS) levels were determined using DCFDA. NDUFA4L2 expression in patients was evaluated by immunohistochemistry, and relapse-free survival was analyzed using the Kaplan–Meier method.Results:NDUFA4L2 was highly expressed in the TR HER2-positive breast cancer cell line. High expression level of NDUFA4L2 was associated with shorter relapse-free intervals in trastuzumab-treated HER2-positive breast cancer patients. Overexpression of NDUFA4L2 enhanced Warburg effects, enhanced aerobic glycolysis, reduced oxygen consumption, and lowered ROS production. Mechanistically, overexpression of NDUFA4L2 facilitated mitochondrial relocalization of HER2 and suppressed ROS production, thus rendering cancer cells more resistant to trastuzumab treatment.Conclusions:We identified NDUFA4L2 as a new biomarker and potential therapeutic target for TR HER2-positive breast cancer.

Construction of an immune-related gene signature to predict survival and treatment outcome in gastric cancer.

Immune cells have emerged as key regulators in the occurrence and development of multiple tumor types. However, it is unclear whether immune-related genes (IRGs) and the tumor immune microenvironment can predict prognosis for patients with gastric cancer (GC). The mRNA expression data in GC tissues (n = 368) were obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed IRGs in patients with GC were determined using a computational difference algorithm. A prognostic signature was constructed using COX regression and random survival forest (RSF) analyses. In addition, datasets related to "gemcitabine resistance" and "trastuzumab resistance" (GSE58118 and GSE77346) were obtained for GEO database, and DEGs associated with drug-resistance were screened. Then, we analyzed correlations between gene expression and cancer immune infiltrates via Tumor Immune Estimation Resource (TIMER) site. The cBioportal database was used to analyze drug-resistant gene mutation status and survival. One hundred and fifty-five differentially expressed IRGs were screened between GC and normal tissues, and a prognostic signature consisting of four IRGs (NRP1, PPP3R1, IL17RA, and FGF16) was closely related to the overall survival (OS). According to cutoff value of risk score, patients were divided into high-risk and low-risk group. Patients in the high-risk group had shorter OS compared to the low-risk group in both the training (p < 0.0001) and testing sets (p = 0.0021). In addition, we developed a 5-IRGs (LGR6, DKK1, TNFRSF1B, NRP1, and CXCR4) signature which may participate in drug resistance processes in GC. Survival analysis showed that patients with drug-resistant gene mutations had shorter OS (p = 0.0459) and DFS (p < 0.001). We constructed four survival-related IRGs and five IRGs related to drug resistance which may contribute to predict the prognosis of GC.

Primary Trastuzumab Resistance After (Neo)adjuvant Trastuzumab-containing Treatment for Patients With HER2-positive Breast Cancer in Real-world Practice

BackgroundIt is difficult to define patients with primary trastuzumab resistance (PTR) after (neo)adjuvant trastuzumab with minimal data and understanding of the actual prevalence, prognosis, and potential treatment strategies in the real-world setting. Patients and MethodsThe medical records of 1096 patients with human epidermal growth factor receptor 2-positive early-stage breast cancer who had received (neo)adjuvant trastuzumab-containing treatment from 2010 to 2016 in the Cancer Hospital and Chinese Academy of Medical Sciences were reviewed. PTR was defined as recurrence during adjuvant trastuzumab or within 12 months from their last (neo)adjuvant trastuzumab dosage. The cutoff date for data collection was September 1, 2018, with a median follow-up time of 46 months. ResultsA total of 126 recurrences were observed, and 75 (6.8%; 75/1096) of them were categorized as PTR; the remaining were non-PTR. The prognosis of patients in the PTR group was much inferior to those in the non-PTR group (27 months vs. not reached; P < .01). As expected, patients with PTR did possess a much lower response rate to first-line trastuzumab-containing therapy (27.3% vs. 61.9%; P = .02). Subgroup analyses indicated that patients in the PTR group seemed to get little survival benefit from the reuse of trastuzumab compared with those without trastuzumab (26 months vs. 28 months; P = .80). However, in the non-PTR group, re-treatment with trastuzumab in the metastatic setting prolonged the survival of patients compared to those without trastuzumab (not reached vs. 34 months; P = .04). ConclusionThis study verified the rationality of present definition of PTR after (neo)adjuvant trastuzumab. Patients with PTR did have a poor prognosis. Further research and clinical trials are required to establish the best treatment patterns for these patients.

MSC-induced lncRNA AGAP2-AS1 promotes stemness and trastuzumab resistance through regulating CPT1 expression and fatty acid oxidation in breast cancer.

Trastuzumab resistance has been becoming a major obstacle for treatment of HER-2-positive breast cancer patients. Increasing evidence suggests that mesenchymal stem cells (MSCs) play critical roles during the formation of drug resistance, however, the underlying mechanism is not well known. In this study, mass spectrometry, RNA pulldown and RNA immunoprecipitation assays were performed to verify the direct interactions among AGAP2-AS1 and other associated targets, such as human antigen R (HuR), miR-15a-5p, and carnitine palmitoyl transferase 1 (CPT1). In vitro and in vivo experimental assays were done to clarify the functional role of AGAP2-AS1 in trastuzumab resistance, stemness, and fatty acid oxidation (FAO). The results showed that MSC co-culture induced trastuzumab resistance. AGAP2-AS1 was upregulated in MSC-cultured cells, and knockdown of AGAP2-AS1 reversed the MSC-mediated trastuzumab resistance. Furthermore, MSC culture-induced AGAP2-AS1 regulates stemness and trastuzumab resistance via activating FAO. Mechanistically, AGAP2-AS1 is associated with HuR, and the AGAP2-AS1-HuR complex could directly bind to the CPT1, increasing its expression via improving RNA stability. In addition, AGAP2-AS1 could serve as ceRNA via sponging miR-15a-5p and releasing CPT1 mRNA. Clinically, increased expression of serum AGAP2-AS1 predicts poor response to trastuzumab treatment in breast cancer patients. In conclusion, MSC culture-induced AGAP2-AS1 caused stemness and trastuzumab resistance via promoting CPT1 expression and inducing FAO. Our results provide new insight of the role of MSCs in trastuzumab resistance and AGAP2-AS1 could be promising predictive biomarker and therapeutic target for HER-2+ breast cancer patients.

Clinical Significance of ARID1A and ANXA1 in HER-2 Positive Breast Cancer.

Background: trastuzumab is considered the standard of care for human epidermal growth factor receptor-2 (HER-2+) breast cancer patients. Regardless of the benefits of its use, many early-stage patients eventually recur, and usually, the disease progresses within a year. Since about half of the HER-2+ patients do not respond to trastuzumab, new biomarkers of prognosis and prediction are warranted to allow a better patient stratification. Annexin A1 (ANXA1) was previously reported to contribute to trastuzumab resistance through AKT activation. An association between adenine thymine-rich interactive domain 1A (ARID1A) loss and ANXA1 upregulation was also previously suggested by others. Methods: in this study, we examined tissue samples from 215 HER-2+ breast cancer patients to investigate the value of ARID1A and ANXA1 protein levels in trastuzumab response prediction and patient outcome. Expression of ARID1A and ANXA1 were assessed by immunohistochemistry. Results: contrary to what was expected, no inverse association was found between ARID1A and ANXA1 expression. HER-2+ (non-luminal) tumours displayed higher ANXA1 expression than luminal B-like (HER-2+) tumours. Concerning trastuzumab resistance, ARID1A and ANXA1 proteins did not demonstrate predictive value as biomarkers. Nevertheless, an association was depicted between ANXA1 expression and breast cancer mortality and relapse. Conclusions: overall, our results suggest that ANXA1 may be a useful prognostic marker in HER-2+ patients. Additionally, its ability to discriminate between HER-2+ (non-luminal) and luminal B-like (HER-2+) patients might assist in patient stratification regarding treatment strategy.

50P Efficacy and safety analysis of pyrotinib in lapatinib resistant HER2-positive metastatic breast cancer: A retrospective study

Lapatinib has shown effectiveness in treating HER2-positive metastatic breast cancer, but therapies after lapatinib resistance are still controversial. In this retrospective study, we assessed the efficacy and safety of pyrotinib in lapatinib resistant HER2-positive metastatic breast cancer. From August 2018 to March 2020, 76 HER2-positive metastatic breast cancer patients who previously failed by lapatinib received pyrotinib in four hospitals. The primary endpoint was investigator-assessed progression-free survival (PFS) per Respond Evaluation Criteria in Solid Tumors (RECIST) version 1.1. The secondary endpoint was the overall survival (OS) and safety of pyrotinib. 66 (86.8%) patients received pyrotinib immediately after lapatinib and 10 (13.2%) received pyrotinib following one or more other therapies. The median PFS of pyrotinib was 8.0 months (95%CI 5.1-10.9) and OS has not reached. Objective response rate (ORR) was 17.1%, and clinical benefit rate (CBR) was 60.5%. Patients who benefited from lapatinib ≥6.0 months were found to have a longer PFS (P=0.034; stratified hazard ratio [HR] 0.534, 95%CI 0.293-0.975). In patients who had received lapatinib in 3 or later line therapy (35, 46.1%), the median PFS of pyrotinib was 9.9 months (95%CI 6.97-12.83) and was relevant to whether lapatinib PFS had reached 6.0 months (P=0.044; HR 0.412, 95%CI 0.167-1.013). No relations were detected between pyrotinib PFS and estrogen receptor (ER) status, trastuzumab resistance, brain metastasis or the sequential use of pyrotinib. In patients who had received lapatinib earlier (41,53.9%), the median PFS of pyrotinib was 6.4 months (95%CI 3.57-9.23). No relevant factors were observed. There was no difference in PFS between these two groups with different lapatinib lines. Toxicity profiles were similar in both groups. The most common adverse effects were diarrhea (34, 44.7%) and hand-foot syndrome (10, 13.2%). Pyrotinib could improve the survival of HER2-positive metastatic breast cancer patients after the failure of lapatinib. For patients who benefited from lapatinib ≥ 6.0 months in 3 or later line therapy, pyrotinib could provide a clinically meaningful longer PFS.

Molecular landscape and efficacy of HER2-targeted therapy in patients with HER2-mutated metastatic breast cancer

Human epidermal growth factor receptor 2 (HER2) protein overexpression or gene amplification is an important predictive biomarker for identifying patients with breast cancer, who may benefit from HER2-targeted therapy. However, little is known about the molecular landscape and efficacy of HER2-targeted therapy in patients with HER2-mutated metastatic breast cancer. We analysed the HER2 mutation features of 1184 patients with invasive breast cancer. In addition, a single-arm, prospective, phase-II study (NCT03412383) of pyrotinib was conducted in patient with metastatic HER2 amplification-negative, mutation-positive breast cancer. Peripheral blood was collected from each patient and circulating tumour DNA (ctDNA) sequencing was performed using a 1021 gene panel. HER2 mutations were detected in 8.9% (105/1184) of patients. The HER2 amplification-positive patients had a higher mutation frequency than the HER2 amplification-negative patients (19.5% vs. 4.8%, P < 0.001). A multivariate Cox regression analysis indicated that patients with HER2 mutations had a shorter progression-free survival (PFS) than HER2 wild-type patients (median PFS 4.7 months vs. 11.0 months, hazard ratio 2.65, 95% confidence interval 1.25–5.65, P = 0.011). Ten HER2 amplification-negative, mutation-positive patients who received pyrotinib monotherapy were ultimately included in the efficacy analysis. The median PFS was 4.9 months. The objective response rate (complete response + partial response) was 40.0% and the clinical benefit rate (complete response + partial response + stable disease over 24 weeks) was 60%. In conclusion, a HER2 gene mutation analysis is potentially useful to identify biomarkers of trastuzumab resistance in HER2 amplification-positive patients. Patients with HER2-mutated, non-amplified metastatic breast cancers may benefit from pyrotinib.

Cyclin E overexpression confers resistance to trastuzumab through noncanonical phosphorylation of SMAD3 in HER2+ breast cancer

ABSTRACT The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-β signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank). In vitro studies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p < .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.

Establishment of a type II insulin-like growth factor receptor gene site-integrated SKBR3 cell line using CRISPR/Cas9.

Human epidermal growth factor receptor 2 (HER-2)+ breast cancer has a high recurrence rate and a poor prognosis, with drug resistance contributing to disease progression. The present study aimed to establish a SKBR3 cell line with type II insulin-like growth factor receptor (IGR-IIR) gene site integration using the CRISPR/Cas9 system, and to provide a cell model for exploring the mechanism responsible for the effect of IGF-IIR on trastuzumab resistance in HER-2+ breast cancer cells. In the present study, six single guide (sg)RNA pairs according to the adeno-associated virus integration site 1 (AAVS1) gene sequence were designed and synthesized, and the Universal CRISPR Activity assay CRISPR/Cas9 rapid construction and activity detection kit was used to connect the annealed oligo with the pCS vector. The sgRNA with the highest efficiency was selected to construct a Cas9/sgRNA expression vector using AsiSI + Bstz17I restriction enzymes to cut IGF-IIR. The fragment was ligated into an human AAVS1-KI vector to construct the IGF-IIR targeting vector. The Cas9/sgRNA and IGF-IIR targeting vectors were electroporated into SKBR3 cells, screened using puromycin and identified via PCR, and the mixed cloned cells generated via IGF-IIR gene targeted integration were obtained. The semi-solid and limited dilution methods were used for monoclonal cell preparation, and the results revealed that a Cas9/sgRNA vector that targeted the AAVS1 was successfully constructed. sgRNA activity detection demonstrated that sgRNA2 had the highest efficiency, while enzyme digestion and sequencing confirmed that the IGF-IIR target vector was successfully constructed. The optimum conditions for electrotransfection were 1,200 V, 20 ms and 2 pulses, and the optimal screening concentration of puromycin was 0.5 µg/ml. Using these conditions, the IGF-IIR targeting vector and pCS-sgRNA2 plasmid were successfully transfected into SKBR3 cells, and PCR identification and sequencing verified the correct genotype of mixed clone fragments. The monoclonal cells proliferate slowly and gradually underwent apoptosis. Overall, the present study successfully obtained a mixed clone cell line with site-specific integration of the IGF-IIR gene at the AAVS1.

Trastuzumab-induced upregulation of a protein set in extracellular vesicles emitted by ErbB2-positive breast cancer cells correlates with their trastuzumab sensitivity

BackgroundErbB2/HER2 oncoprotein often drives breast cancers (BCs) which are treated with the anti-ErbB2 antibody trastuzumab. The efficacy of trastuzumab-based metastatic BC therapies is routinely assessed by imaging studies. Trastuzumab typically becomes ineffective in the case of this disease and is then replaced by other drugs. Biomarkers of BC trastuzumab response could allow imaging studies and the switch to other drugs to occur earlier than is now possible. Moreover, bone-only BC metastases can be hard to measure, and biomarkers of their trastuzumab response could facilitate further treatment decisions. Such biomarkers are presently unavailable. In this study, we searched for proteins whose levels in BC cell-emitted extracellular vesicles (EVs) potentially correlate with BC trastuzumab sensitivity.MethodsWe isolated EVs from cultured trastuzumab-sensitive and trastuzumab-resistant human BC cells before and after trastuzumab treatment and characterized these EVs by nanoparticle tracking analysis and electron microscopy. We found previously that ErbB2 drives BC by downregulating a pro-apoptotic protein PERP. We now tested whether trastuzumab-induced PERP upregulation in EVs emitted by cultured human BC cells correlates with their trastuzumab sensitivity. We also used mass spectrometry to search for additional proteins whose levels in such EVs reflect BC cell trastuzumab sensitivity. Once we identified proteins whose EV levels correlate with this sensitivity in culture, we explored the feasibility of testing whether their levels in the blood EVs of trastuzumab-treated metastatic BC patients correlate with patients’ response to trastuzumab-based treatments.ResultsWe found that neither trastuzumab nor acquisition of trastuzumab resistance by BC cells affects the size or morphology of EVs emitted by cultured BC cells. We established that EV levels of proteins PERP, GNAS2, GNA13, ITB1, and RAB10 correlate with BC cell trastuzumab response. Moreover, these proteins were upregulated during trastuzumab-based therapies in the blood EVs of a pilot cohort of metastatic BC patients that benefited from these therapies but not in those derived from patients that failed such treatments.ConclusionsUpregulation of a protein set in EVs derived from cultured breast tumor cells correlates with tumor cell trastuzumab sensitivity. It is feasible to further evaluate these proteins as biomarkers of metastatic BC trastuzumab response.

LncRNA-SNHG7 Enhances Chemotherapy Resistance and Cell Viability of Breast Cancer Cells by Regulating miR-186.

BackgroundClinical tolerance to trastuzumab greatly affects the therapeutic effect in breast cancer (BC). Long-chain non-coding RNA (lncRNA) plays an important role in the development of trastuzumab resistance, in which SNHG7 can promote the epithelial mesenchymal transformation (EMT) of breast cancer cells into, while EMT is related to trastuzumab resistance of breast cancer cells.ObjectiveTo investigate whether lncRNA-SNHG7 can enhance chemotherapy resistance and cell viability of BC cells by regulating miR-186.MethodsSK-BR-3 and SNHG7 of HER2+BC cells were induced to enhance the resistance of BC cells to trastuzumab by regulating miR-186, and to regulate the expression levels of SNHG7 and miR-186. The sensitivity of drug-resistant cells to trastuzumab and the changes of cell proliferation, migration, apoptosis, and EMT were measured and verified by tumorigenesis in vivo. The effects of miR-186 on SNHG7 were investigated through rescue experiments; the regulatory relationship between the expression of SNHG7 and miR-186 was verified by the double luciferase reporter (DLR) and the mechanism of SNHG7 was explored.ResultsDown-regulation of SNHG7 or up-regulation of miR-186 could increase the sensitivity of BC cells to trastuzumab, inhibit the proliferation, migration and EMT, and promote apoptosis. Compared with the down-regulation of SNHG7 or miR-186 alone, simultaneous down-regulation of SNHG7 and miR-186 on drug-resistant cells brought notably lower sensitivity to trastuzumab and apoptosis rate, and higher proliferation and apoptosis ability. The DLR showed that miR-186 could specifically inhibit the expression of SNHG7. The results of tumorigenesis in vivo revealed that down-regulation of SNHG7 or up-regulation of miR-186 could improve the therapeutic effect of trastuzumab and reduce the tumor volume, and miR-186 could also antagonize the effect of SNHG7.ConclusionDown-regulation of SNHG7-targeted miR-186 can reverse trastuzumab resistance of BC cells, inhibit the proliferation, migration, and EMT levels of BC cells, and promote apoptosis.

POU4F1 confers trastuzumab resistance in HER2-positive breast cancer through regulating ERK1/2 signaling pathway

Over-expression of the human epidermal growth factor receptor-2 (HER2) is related to aggressive tumors and poor prognosis in breast cancer. Trastuzumab (TRA) resistance leads to tumor recurrence and metastasis, resulting in poor prognosis in HER2-positive breast cancer. POU Class 4 Homeobox 1 (POU4F1) is a member of the POU domain family transcription factors, and has a key role in regulating cancers. However, its effects on TRA-resistant HER2-positive breast cancer are still vague. In the present study, we found that POU4F1 expression was dramatically increased in clinical breast cancer specimens with TRA resistance. Higher POU4F1 was also detected in HER2-positive breast cancer cells with TRA resistance than that of the parental ones. Poor prognosis was detected in breast cancer patients with high POU4F1 expression. Under TRA treatment, POU4F1 knockdown significantly reduced the proliferative capacity of HER2-positive breast cancer cells with TRA resistance. POU4F1 silence also sensitized resistant HER-positive breast cancer cells to TRA treatment in vivo using a xenograft mouse model, along with the markedly reduced tumor growth rate and tumor weight. Moreover, we found that POU4F1 deletion greatly decreased the activation of mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2 (MEK1/2) and extracellular-regulated kinase 1/2 (ERK1/2) signaling pathways in breast cancer cells with TRA resistance. Migration and invasion were also effectively hindered by POU4F1 knockdown in TRA-resistant HER2-positive breast cancer cells. Notably, we found that POU4F1 deletion-improved chemosensitivity of HER2-positive breast cancer cells with drug-resistance to TRA treatment was closely associated with the blockage of ERK1/2 signaling. Collectively, our findings reported a critical role of POU4F1 in regulating TRA resistance, and demonstrated the underlying molecular mechanisms in HER2-positive breast cancer. Thus, POU4F1 may be a promising prognostic and therapeutic target to develop effective treatment for overcoming TRA resistance.

The Hippo Transducer YAP/TAZ as a Biomarker of Therapeutic Response and Prognosis in Trastuzumab-Based Neoadjuvant Therapy Treated HER2-Positive Breast Cancer Patients.

BackgroundWe explored the therapeutic and prognostic effect of YAP/TAZ intensityinHER2-positive breast cancer patients. We also investigated the relationship between YAP/TAZ expression and Trastuzumab-resistance.MethodsWe collected clinicopathological information from 397 cases. We evaluated therapeutic and prognostic effect of YAP/TAZ and other variables. We also cultivated Trastuzumab-resistance cell lines and explored relationship between YAP/TAZ and Trastuzumab-resistance.ResultsOver-expression of YAP/TAZ was remarkable in Trastuzumab-resistant cells, and so did HER3 and HER2/HER3 heterodimer. Inhibition of YAP/TAZ expression reversed Trastuzumab-resistance.YAP/TAZ deficiency contributed to favorable therapeutic response, and so did hormone receptor insufficiency and chemotherapy dosage inferiority. Deficient YAP/TAZ intensity and abundant hormone receptor intensity contributed to better survival. Over-expression of YAP/TAZ was obvious in recurrent cases in comparison with their matching primary lesions. Prognostic superiority of insufficient YAP/TAZ intensity was more outstanding in hormone receptor negative cases. Over-expression of YAP/TAZ and HER3 was generally synchronous. Absence of HER3 expression in residual lesions might correlate with better breast cancer-free survival.ConclusionsOver-expression of YAP/TAZ as well as HER-3 and HER2/HER3 heterodimer was synchronously remarkable in Trastuzumab-resistant cell lines. Inhibition of YAP/TAZ expression reversed Trastuzumab resistance. Deficient YAP/TAZ intensity as well as insufficient hormone receptor intensity and high chemotherapy dosage contributed to favorable therapeutic response. Deficient YAP/TAZ intensity and abundant hormone receptor intensity contributed to better survival, and so did absence of HER3expression in residual lesions. Prognostic superiority of YAP/TAZ expression depended on hormone receptor status. Cases with synchronous over-expression of YAP/TAZ and HER3 suffered poor survival, which revealed the potential effect of YAP/TAZ-HER2/HER3 crosstalk in prognosis of HER2-positive patients.

A novel rabbit derived anti-HER2 antibody with pronounced therapeutic effectiveness on HER2-positive breast cancer cells in vitro and in humanized tumor mice (HTM)

BackgroundAntibody based cancer therapies have achieved convincing success rates combining enhanced tumor specificity and reduced side effects in patients. Trastuzumab that targets the human epidermal growth factor related receptor 2 (HER2) is one of the greatest success stories in this field. For decades, trastuzumab based treatment regimens are significantly improving the prognosis of HER2-positive breast cancer patients both in the metastatic and the (neo-) adjuvant setting. Nevertheless, ≥ 50% of trastuzumab treated patients experience de-novo or acquired resistance. Therefore, an enhanced anti-HER2 targeting with improved treatment efficiency is still aspired.MethodsHere, we determined cellular and molecular mechanisms involved in the treatment of HER2-positive BC cells with a new rabbit derived HER2 specific chimeric monoclonal antibody called “B100″. We evaluated the B100 treatment efficiency of HER2-positive BC cells with different sensitivity to trastuzumab both in vitro and in the presence of a human immune system in humanized tumor mice.ResultsB100 not only efficiently blocks cell proliferation but more importantly induces apoptotic tumor cell death. Detailed in vitro analyses of B100 in comparison to trastuzumab (and pertuzumab) revealed equivalent HER2 internalization and recycling capacity, similar Fc receptor signaling, but different HER2 epitope recognition with high binding and treatment efficiency. In trastuzumab resistant SK-BR-3 based humanized tumor mice the B100 treatment eliminated the primary tumor but even more importantly eradicated metastasized tumor cells in lung, liver, brain, and bone marrow.ConclusionOverall, B100 demonstrated an enhanced anti-tumor activity both in vitro and in an enhanced preclinical HTM in vivo model compared to trastuzumab or pertuzumab. Thus, the use of B100 is a promising option to complement and to enhance established treatment regimens for HER2-positive (breast) cancer and to overcome trastuzumab resistance. Extended preclinical analyses using appropriate models and clinical investigations are warranted.

Abstract 1915: Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling

Abstract Introduction The widespread clinical use of monoclonal antibodies such as trastuzumab in personalized medicine, targeting upstream proteins such as receptors with cascading effects on downstream signaling, elicits the importance of studying the phosphoproteome in the investigation of resistance to targeted therapy. This study sought to differentiate between the complex mechanisms of intrinsic and acquired trastuzumab resistance, by quantifying changes in expression and activity of proteins in key signal transduction pathways, in cellular models of resistance. Method DigiWest®, a Luminex® xMAP® bead-based high-throughput protein profiling method, was utilized to measure 194 proteins and phospho-proteins from 12µg of total protein, obtained from HER2-positive cell lines which were trastuzumab-sensitive (SKBR3), intrinsically resistant (JIMT-1, UACC-893) and with acquired resistance (T15, obtained by long-term culture of SKBR3 in 15µg/mL trastuzumab). The resulting median fluorescence intensities (MFI) were normalized to total protein content. Relative quantification was done against SKBR3 and a log2 fold-change of ±1 was considered to be significant. For each of the three trastuzumab-resistant cell lines, targets which were differentially expressed were organized into established signal transduction pathways, and specific phosphosite expression values were used to determine whether each protein was under- or over-activated. Results A total of 115 proteins were expressed in at least one of the HER2-positive cell lines at a normalized MFI above the set threshold. JIMT-1 showed the most concurrent resistance mechanisms, including PI3K/Akt and RAS/RAF/MEK/ERK activation, β-catenin stabilization, cell cycle progression by Rb suppression, and CREB-mediated cell survival. MAPK (ERK) pathway activation was common to both intrinsic and acquired resistance – it was over-activated in all the three trastuzumab-resistant cell lines. The overexpression of upstream RAS, however, was confined to the intrinsically resistant cell lines JIMT-1 and UACC-893; meanwhile, in T15, entry into the ERK pathway was mostly mediated by pan-PKC overexpression. PKCs, especially PKCα, were also highly activated in UACC-893. β-catenin stabilization was another mechanism observed only in the intrinsically trastuzumab-resistant cell lines. Conclusion Both distinct and overlapping mechanisms are involved in intrinsic and acquired trastuzumab resistance. Multiple pathways can also co-exist to promote cell survival, as was mostly exemplified by JIMT-1, derived from an aggressive, basal-like tumor. Furthermore, different models of intrinsic resistance (JIMT-1 vs UACC-893) showed significant differences in differentially activated signal transduction pathways. DigiWest® provided a simple and robust multiplexing solution for this phospho-proteomic study. Citation Format: Jeanesse Scerri, Felix Ruoff, Markus Templin, Godfrey Grech, Christian Scerri. Intrinsic and acquired trastuzumab resistance pathways in breast cancer cell lines using DigiWest® phospho-protein profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1915.

Abstract 5295: Impact of a multi-gene expression profile in predicting adjuvant trastuzumab outcome in HER2-positive breast cancer

Abstract Background Trastuzumab is the cornerstone of adjuvant therapy for HER2-positive breast cancer (BC), but up to 20% of patients relapse. Understanding resistance mechanisms could allow developing strategies to overcome this issue. We performed a case-control, retrospective study to compare the gene expression profiles (GEPs) of 27 patients who experienced disease relapse within 2 years from the start of adjuvant trastuzumab therapy, with those of 27 patients who were disease-free after at least 5 years. Here, we present the preliminary results on 12 cases and 12 controls, matched by age and hormone receptor status. Methods RNA was isolated from formalin-fixed paraffin-embedded primary tumors (FFPE) with AllPrep DNA/RNA FFPE Kit (Qiagen) and quantified by Nanodrop, before performing Nanostring gene expression profile. nCounter Breast Cancer 360 Panel (Nanostring Technologies) was used according to manufacturer's instructions. nSolver analysis software (Nanostring Technologies) was used to obtain normalized counts and to compare GEPs between cases and controls, considering as significant a Benjamini Yekutieli (BY) corrected p-value ≤0.05. Mann-Whitney median test was used to compare Ki67 score and Body Mass Index (BMI) between cases and controls. Results Out of 770 analyzed genes, none was significantly differentially expressed at BY p-value &amp;lt;0.05, probably due to the low number of cases. Five genes were differentially expressed with a BY p-value &amp;lt;0.1: CDKN1A (Log2 fold change -0.733, 95% CI: -1.06 − -0.409), GDF15 (Log2 fold change -1.92, 95% CI: -2.78 − -1.05), TFDP1 (Log2 fold change -0.567, 95% CI: -0.826 − -0.309) and ELF3 (Log2 fold change -1.52, 95%CI: -2.22 − -0.813) were downregulated in control patients, whereas TNFSF10 (Log2 fold change 1.27, 95% CI: 0.669 − 1.88) was upregulated. CDKN1A and TFDP1 are cell cycle regulators: the former encodes for p21, which inhibits cell cycle progression and is involved in DNA damage repair; the latter encodes a transcription factors that dimerizes with E2F, promoting cell cycle genes' transcription. GDF15 codes for a secreted ligand of the TGF-beta superfamily and acts as a pleiotropic cytokine involved in response to hypoxia, inflammation, acute injury and oxidative stress. ELF3 is a transcriptional activator involved in vascular inflammation and a critical downstream effector of HER2 pathway. TNFSF10 encodes a cytokine belonging to the TNF ligand family, inducing apoptosis in tumor cells. No differences were found between cases and controls considering Ki67 proliferation index and BMI. Conclusions Our preliminary results indicate that altered expression of genes involved in cell cycle, apoptosis, inflammation and stress response are involved in trastuzumab resistance. The validation of these results overall series is ongoing. The relations between GEPs and clinico-pathological features will be assessed. Citation Format: Sara Ravaioli, Roberta Maltoni, Francesca Pirini, Michela Palleschi, William Balzi, Elisabetta Petracci, Giovanni Martinelli, Andrea Rocca, Sara Bravaccini. Impact of a multi-gene expression profile in predicting adjuvant trastuzumab outcome in HER2-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5295.

Abstract 4222: Tucatinib, a selective small molecule HER2 inibitor, is active in HER2 mutant driven tumors

Abstract Tucatinib is an investigational, orally dosed, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2 without significant inhibition of EGFR. In this report, we describe the activity of tucatinib alone and in combination with trastuzumab in multiple HER2 mutant driven PDX models. Tucatinib was tested in 6 HER2 mutant PDX tumor models derived from colorectal, non-small cell lung cancer, gall bladder and gastric cancers with L755S, V77L or S310Y mutations. MCF-10A cells were engineered to express the L755S and V777L HER2 activating point mutations and the potency of tucatinib was evaluated in each of these cell lines using HER2 autophosphorylation as an endpoint. The activity of tucatinib was also evaluated in G776V_G/C and G776_YVMA in frame exon 20 insertion mutations, including the G776V_G/C mutant non-small cell lung cancer line NCI-H1781 using both cellular signaling and cell proliferation as endpoints. Tucatinib significantly inhibited tumor growth in 5 of the 6 HER2 point mutant PDX models, whereas trastuzumab alone significantly inhibited tumor growth in 1 of 6 models and the combination of both drugs was significantly active in all HER2 mutant models. Tucatinib inhibited the phosphorylation of HER2 in a L755S mutant MCF-10A cell line and inhibited the growth of 3 L755S mutant tumor models derived from colorectal, gastric and non-small cell lung cancer. Tucatinib also potently inhibited the phosphorylation of HER2 in a V777L mutant MCF-10A cell line, and in a colorectal PDX model containing a V777L mutation, tucatinib alone or in combination with trastuzumab induced tumor regressions. In two PDX models containing the S310Y extracellular domain mutation of HER2, tucatinib alone, or in combination with trastuzumab, resulted in tumor growth delays. In exon 20 insertion mutant derived cell lines, tucatinib inhibited HER2 phosphorylation, but not EGFR phosphorylation, and inhibited the phosphorylation of HER2, HER3, Erk1/2, MEK1 and AKT in the G776V_G/C HER2 mutant NCI-H1781 cell line. These data demonstrate that tucatinib, a highly potent and selective inhibitor of HER2 signaling without significant inhibition of EGFR, is active in multiple HER2 mutant driven tumor models. Importantly, tucatinib is active in models containing the L755S HER2 mutation which has been associated with lapatinib resistance preclinically (1), and trastuzumab resistance clinically (2). Taken together, these results support the idea that HER2 selective inhibition by tucatinib can block oncogenic signaling elicited by mutant HER2, and that it could potentially provide clinical benefit in patients with cancers with activating HER2 mutations.

Therapeutic Strategies for Human Epidermal Receptor-2 Positive Metastatic Breast Cancer: A Literature Review.

Breast cancer is a frequently occurring malignancy in women. Immunologically, breast cancers can be classified into four subtypes depending on the types of receptors present and their expression profiles. These are estrogen positive, progesterone positive, human epidermal growth factor receptor type 2 (HER2) positive, and triple-negative as identified by immunohistochemistry. This classification is the basis of response to treatment, prognosis, and survival. With the identification of HER2 receptor overexpression, targeted therapies with anti-HER2 agents have been developed. The first-line therapy approved for HER2 positive tumors is trastuzumab and pertuzumab linked to taxane and further treatment with an antibody-drug conjugate to achieve satisfactory outcomes. Tyrosine kinase overexpression can be treated with lapatinib, which has also been approved for improving survival and is used in combination with capecitabine. Acquired resistance in HER2 positive tumors is shown in many cases due to genetic or epigenetic modifications. Therefore, it is very important to plan therapeutic strategies and design effective treatment approaches. For a long time, only two agents, trastuzumab and lapatinib, have been approved by the Food and Drug Administration (FDA) for the treatment of HER2 positive breast cancers. There has been no appropriate treatment for trastuzumab resistance and its failure to reduce tumor growth. Lapatinib was approved by the FDA in 2007 for HER2 positive breast cancer. Three existing therapy options after trastuzumab resistance was proposed by clinicians: continuation of trastuzumab, starting therapy with lapatinib, and the synergistic use of trastuzumab and lapatinib.There have been several effective therapies proposed for HER2 positive breast cancers in correlation with clinical trials. Discovering the mechanisms of trastuzumab resistance would increase its response to therapy and better clinical outcome. Clinicians are being continuously challenged by the resistance mechanisms and bioavailability of the drugs in the treatment of metastatic breast cancers. The addition of new drugs to the chemotherapeutic regimen increases the complexity, burden of side effects, and chances of relapse. Novel anti-HER2 agents have been directed towards therapy making a major paradigm shift.