Lapatinib-resistant Cells Research Articles

PI3K PROTAC overcomes the lapatinib resistance in PIK3CA-mutant HER2 positive breast cancer

Although anti-HER2 therapy has made significant strides in reducing metastasis and relapse in HER2-positive breast cancer, resistance to agents like trastuzumab, pertuzumab, and lapatinib frequently develops in patients undergoing treatment. Previous studies suggest that the hyperactivation of the PI3K-AKT signaling pathway by PIK3CA/PTEN gene mutations is implicated in HER2 resistance. In this study, we introduce a novel PI3K-p110α Proteolysis TAargeting Chimera (PROTAC) that effectively inhibits the proliferation of breast cancer cells by degrading PI3K-p110α. When tested in two lapatinib-resistant cell lines, JIMT1 and MDA-MB-453, both of which harbor PIK3CA mutations, the PI3K PROTAC notably reduced cell proliferation and induced G1 phase cell cycle arrest. Importantly, even at very low concentrations, PI3K PROTAC restored sensitivity to lapatinib. Furthermore, the efficacy of PI3K PROTAC surpassed that of Alpelisib, a selective PI3K-p110α kinase inhibitor in clinic. The superior performance of PI3K PROTAC was also confirmed in lapatinib-resistant breast cancer xenograft tumors and patient-derived breast cancer organoids (PDOs). In conclusion, this study reveals that the novel PI3K PROTAC we synthesized could serve as an effective agent to overcome lapatinib resistance.

Expand available targets for CAR-T therapy to overcome tumor drug resistance based on the "Evolutionary Traps"

Based on the concept of "Evolutionary Traps", targeting survival essential genes obtained during tumor drug resistance can effectively eliminate resistant cells. While, it still faces limitations. In this study, lapatinib-resistant cells were used to test the concept of "Evolutionary Traps" and no suitable target stand out because of the identified genes without accessible drug. However, a membrane protein PDPN, which is low or non-expressed in normal tissues, is identified as highly expressed in lapatinib-resistant tumor cells. PDPN CAR-T cells were developed and showed high cytotoxicity against lapatinib-resistant tumor cells in vitro and in vivo, suggesting that CAR-T may be a feasible route for overcoming drug resistance of tumor based on "Evolutionary Trap". To test whether this concept is cell line or drug dependent, we analyzed 21 drug-resistant tumor cell expression profiles reveal that JAG1, GPC3, and L1CAM, which are suitable targets for CAR-T treatment, are significantly upregulated in various drug-resistant tumor cells. Our findings shed light on the feasibility of utilizing CAR-T therapy to treat drug-resistant tumors and broaden the concept of the "Evolutionary Trap".

Artemisia argyi extracts overcome lapatinib resistance via enhancing TMPRSS2 activation in HER2-positive breast cancer.

Breast cancer stands as the predominant malignancy and primary cause of cancer-related mortality among females globally. Approximately 25% of breast cancers exhibit HER2 overexpression, imparting a more aggressive tumor phenotype and correlating with poor prognoses. Patients with metastatic breast cancer receiving HER2 tyrosine kinase inhibitors (HER2 TKIs), such as Lapatinib, develop acquired resistance within a year, posing a critical challenge in managing this disease. Here, we explore the potential of Artemisia argyi, a Chinese herbal medicine known for its anti-cancer properties, in mitigating HER2 TKI resistance in breast cancer. Analysis of the Cancer Genome Atlas (TCGA) revealed diminished expression of transmembrane serine protease 2 (TMPRSS2), a subfamily of membrane proteolytic enzymes, in breast cancer patients, correlating with unfavorable outcomes. Intriguingly, lapatinib-responsive patients exhibited higher TMPRSS2 expression. Our study unveiled that the compounds from Artemisia argyi, eriodictyol, and umbelliferone could inhibit the growth of lapatinib-resistant HER2-positive breast cancer cells. Mechanistically, they suppressed HER2 kinase activation by enhancing TMPRSS2 activity. Our findings propose TMPRSS2 as a critical determinant in lapatinib sensitivity, and Artemisia argyi emerges as a potential agent to overcome lapatinib via activating TMPRSS2 in HER2-positive breast cancer. This study not only unravels the molecular mechanisms driving cell death in HER2-positive breast cancer cells induced by Artemisia argyi but also lays the groundwork for developing novel inhibitors to enhance therapy outcomes.

Abstract LB228: Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells

Abstract Background: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinases, which is approved for HER2-positive metastatic breast cancer as second line treatment. Significant proportion of patients experience disease progression due to acquired resistance. Activation of DNA damage repair (DDR) is one of the drug-resistance mechanism, however, the impact of DDR on sensitivity to lapatinib is unclear. Thus, lapatinib resistance mechanism was explored from the standpoint of DDR activation. Methods: Acquired lapatinib-resistant (LR) SK-BR-3 cell lines were generated by continuously exposing to lapatinib, starting with 30 nmol/L and incrementally increasing to 10 μmol/L over 7 months. MTT assay was used to confirm lapatinib sensitivity. Cell cycle progression was analyzed using BrdU assay. Replication fork speed and stalled fork were analyzed by DNA fiber assay. Expression of the molecules was examined using Western blotting, immunofluorescence assay and transcriptome data analysis. DNA strand breaks and repair efficacy were evaluated through alkaline comet assay. Results: G1/S phase transition was increased and early/late S phase population was increased in SK-BR-3 LR cells. Replication fork speed was accelerated and replication stress are elevated in SK-BR-3 LR cells. p-Chk1, Rad51, Rad51B, Rad51C and XRCC3 were upregulated in SK-BR-3 LR cells. After irradiation or treatment with ATR inhibitor, γ-H2AX was continuously increased in parental cells. In contrast, γ-H2AX did not change significantly in SK-BR-3 LR cells, and Chk1 was activated after irradiation or treatment with ATR inhibitor. Tail of comet disappeared at early time point in in SK-BR-3 LR cells after induction of DNA damage compared with parental cells. These results demonstrated that DDR was activated and DNA damage repair capacity was enhanced in LR cells. Conclusion: Replication stress is elevated in SK-BR-3 LR cells. Up-regulation of molecules involved in the homologous recombination repair pathway was observed in SK-BR-3 LR cells. Moreover, DNA repair capacity was increased in SK-BR-3 LR cells. These data suggested that activation of DNA damage repair pathway caused by replication stress contributes to lapatinib resistance. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yu Jin Kim, Sujin Ham, Hae Min Hwang, Minyoung Lee, Changyun Lee, Jinyong Kim, Dae-Won Lee, Kyung-Hun Lee, Seock-Ah Im. Replication stress activates the DNA damage response and contributes to lapatinib resistance in HER2-positive SK-BR-3 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB228.

Abstract P2-20-04: Vaccination against HIF1α/c-MET peptides overcome lapatinib resistance and inhibits tumor growth by immunomodulating in HER2 positive breast cancer

Abstract Human Epidermal growth factor Receptor 2 (HER2) is overexpressed in 20% to 30% of breast cancers and is associated with poor prognosis. Patients with advanced HER2-type breast cancer are treated with HER2-directed tyrosine kinase inhibitors as well as antibodies, but many patients finally acquire therapeutic resistance. Therefore, it is important to develop new treatment strategies that circumvent HER2 positive resistant breast cancer. Among the suggested mechanisms of resistance to HER2-targeted therapeutics, tumor hypoxia and relevant biological factors are known as important players. In this study, we aimed to explore the role of tumor hypoxia in the development of resistance to HER2-targeted therapeutics, and whether the role of an immunological therapy targeting this could contribute to overcoming resistance. To this end, we established lapatinib-resistant HER2 positive breast cancer cells in vitro (SKBR3, BT-474 for human and MMC for murine cells). First, we examined if lapatinib-sensitivity is different according to oxygen concentration using lapatinib-resistant cell lines (LR-SKBT3, LR-BT-474, LR-MMC) compared with parent cell lines. Resistant cells were resistant both in normoxic and hypoxic conditions, but parent cells were highly sensitive in both of the oxygen concentration. In the Western blot analysis, HIF1α and c-MET were overexpressed in the resistant cells compared with parent cell lines in both normoxic and hypoxic condition. Interestingly, treatment with lapatinib had reduced HIF1α and c-MET expression at normoxia in parent cell lines but not in resistant cell lines. Under the hypoxic conditions, the difference in expression of HIF1α and c-MET was more pronounced between parent cell lines and resistant cell lines treated with lapatinib. These results indicate that the expression of HIF1α and c-MET was involved in lapatinib-resistant. In a previous study, we have identified epitopes from HIF1α/c-MET and demonstrated effect of delayed bone metastasis in triple negative breast cancer model when used as peptide vaccine. Herein, we evaluated whether the immunization using HIF1α/c-MET peptide vaccine is effective in inhibition of lapatinib-resistant HER2 positive breast cancer growth in vivo. MMTVneu-transgenic mouse model was used and 6- 8 weeks old mice were injected with LR-MMC subcutaneously. Immunization using the HIF1α/c-MET peptide vaccine significantly inhibited growth of LR-MMC cells in MMTVneu-transgenic mice compared with controls. HIF1α/c-MET specific IFN-γ-secreting T cell responses in the splenocytes were significantly higher in the immunized group than controls. Immunohistochemical staining of T cells revealed that expression of tumor HIF1α and c-MET was significantly decreased, while the number of CD8+ T cell was increased in the tumors from immunized animals compared with control tumors. In conclusion, our study demonstrated that the vaccine targeting HIF1α/c-MET might be a promising cancer immunotherapy in lapatinib-resistant HER2 positive breast cancer. In vivo study to evaluate efficacy of combination therapy (lapatinib and HIF1α/c-MET vaccine) is ongoing. Citation Format: Ji Min Lee, Jin Hwa Hong, Soon Young Lim, Ju Won Kim, Ah Reum Lim, Myung Han Hyun, Kyong Hwa Park. Vaccination against HIF1α/c-MET peptides overcome lapatinib resistance and inhibits tumor growth by immunomodulating in HER2 positive breast cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-04.

Abstract LB127: Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells

Abstract The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6) in the metabolic adaptation of rapidly growing versus invasive TNBC cells as well as in Lapatinib resistant (Lap-R) TNBC cells. AnxA6 expression in cultured TNBC cell lines, Lap-R cells and in crude isolates of mitochondria was determined by Western blotting and/or immunofluorescence assays. The oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and mitochondria activity were assessed using the Seahorse XF Analyzer. Intracellular metabolites were detected by 1H-NMR analysis. Using model proliferative basal-like and invasive mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. Down regulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent cellular energy phenotype. We further show that AnxA6-depletion was associated with decreased lipid droplet accumulation and the lipolytic phenotype by enhancing the uptake and mitochondrial fatty acid oxidation for ATP production. Chronic Lapatinib-induced up regulation of AnxA6 in AnxA6-low TNBC cells reversed their lipolytic to a more lipogenic/glycolytic metabolic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies significant metabolic adaptation during stress and/or chronic Lapatinib treatment and provide additional insights into the potential of AnxA6 as a biomarker for not only therapeutic intervention but also the metabolic subtyping of TNBC subsets. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB127.

Reduced Expression of Annexin A6 Induces Metabolic Reprogramming That Favors Rapid Fatty Acid Oxidation in Triple-Negative Breast Cancer Cells.

Simple SummaryThe expression status of Annexin A6 (AnxA6) has been shown to influence tumor growth, metastasis, and resistance to therapeutic intervention, but the contribution of this tumor suppressor in the metabolic adaptation of basal-like (AnxA6-low) versus mesenchymal-like (AnxA6-high) TNBC subsets remains unclear. The downregulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent metabolic phenotype. The overexpression or chronic lapatinib induced the expression of AnxA6 in AnxA6-low TNBC cells and reversed the quiescent phenotype to a more lipogenic/glycolytic phenotype. Interestingly, AnxA6-depletion was associated with rapid fatty acid uptake and oxidation (lypolytic metabolic phenotype) but decreased lipid droplet accumulation. Our data suggest that the expression status of AnxA6 in TNBC cells is associated with distinct metabolic adaptations of basal-like and mesenchymal-like TNBC subsets in response to cellular stress and/or therapeutic intervention.The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6), in the metabolic adaptation of basal-like (AnxA6-low) versus mesenchymal-like (AnxA6-high), as well as in lapatinib-resistant TNBC cells. Using model basal-like and mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. The downregulation of AnxA6 in TNBC cells generally attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity resulting in a quiescent metabolic phenotype. We also show that AnxA6 depletion in mesenchymal-like TNBC cells was associated with a rapid uptake and mitochondrial fatty acid oxidation and diminished lipid droplet accumulation and altered the lipogenic metabolic phenotype of these cells to a lypolytic metabolic phenotype. The overexpression or chronic lapatinib-induced upregulation of AnxA6 in AnxA6-low TNBC cells reversed the quiescent/lypolytic phenotype to a more lipogenic/glycolytic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies distinct metabolic adaptations of basal-like and mesenchymal-like TNBC subsets in response to cellular stress and/or therapeutic intervention and suggest AnxA6 as a biomarker for metabolic subtyping of TNBC subsets.

Induction of EnR stress by Melatonin enhances the cytotoxic effect of Lapatinib in HER2-positive breast cancer.

Despite HER2-targeted cancer treatments have provided considerable clinical benefits, resistance to HER2-targeted agents will inevitably develop. Targeting non-oncogene vulnerabilities including endoplasmic reticulum (EnR) stress has emerged as an attractive alternative approach to improve the efficacy of existing targeted cancer therapies. In the current study, we find that Melatonin sensitizes HER2-positive breast cancer cells to the dual tyrosine kinase inhibitor Lapatinib in vitro. Mechanistically, Melatonin enhances the cytotoxic effects of Lapatinib through promoting excessive EnR stress-induced unfolded protein response (UPR) and ROS overaccumulation. Consistently, the antioxidant N-acetylcysteine remarkably reverses the effects of the drug combination on ROS production, DNA damage and cytotoxicity. Furthermore, Melatonin significantly enhances the anti-tumor effect of Lapatinib in an HCC1954 xenograft model. Meanwhile, Lapatinib resistant HER2-positive breast cancer cells (LapR) display lower basal expression levels of UPR genes and enhanced tolerance to EnR stress with attenuated response to Brefeldin A and Tunicamycin. Importantly, Melatonin also increases the sensitivity of HCC1954 LapR cells to Lapatinib. Together, our findings highlight the potential utility of Melatonin as an adjuvant in the treatment of primary or therapy resistant HER2-positive breast cancer via EnR stress-mediated mechanisms.

Therapeutic Mechanism of Lapatinib Combined with Sulforaphane on Gastric Cancer

Background Lapatinib is a small-molecule tyrosine kinase inhibitor that plays important roles in cell proliferation and survival. Administration of lapatinib with capecitabine is an effective treatment for HER2-positive metastatic BC. However, the effects of lapatinib on gastric cancer (GC) remain to be clear. In this study, we aimed to investigate the therapeutic effects of lapatinib combined with sulforaphane on GC and its underlying mechanisms. Methods SGC-7901 and lapatinib-resistant SGC-7901 cells were treated with lapatinib (0.2 μM), sulforaphane (5 μM), or their combinations. Cell viability, invasion, cycle, and apoptosis of SGC-7901 and lapatinib-resistant SGC-7901 cells were evaluated by thiazolyl blue tetrazolium bromide (MTT), Boyden chamber assay, and flow cytometer. The protein expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were detected by Western blotting. Results We observed that lapatinib combined with sulforaphane significantly decreased cell viability and inhibited cell migration of drug-sensitive and drug-resistant cells. Lapatinib sulforaphane also remarkably induced cell apoptosis with G0/G1 arrest. In addition, Western blotting revealed that the expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were downregulated by lapatinib-sulforaphane treatment. Conclusion Combination of lapatinib and sulforaphane might be a novel and promising therapeutic treatment for lapatinib-sensitive or lapatinib-resistant GC patients.

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA

BackgroundHER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype.MethodsWe generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets.ResultsTreatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts.ConclusionOur results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Strategic co-targeting of FOXM1 and EGFR-ERBB2 signaling in ovarian cancer

Objectives: Epithelial ovarian cancer (EOC) is a challenging disease due to chemoresistance, and short-term recurrence. Chemoresistance to anti-cancer drugs substantially reduces survival in EOC. In this study, we showed that chemoresistance to lapatinib (a dual EGFR-ERBB2 kinase inhibitor) and thiostrepton (FOXM1 inhibitor), induced the epithelial-mesenchymal transition and stemness in OC cells. Aberrant expression of FOXM1 and EGFR-ERBB2 proteins are common in many solid cancers including serous EOC. However, the precise mechanism of FOXM1, EGFR-ERBB2 in the chemoresistance of OC remains unclear. Methods: We blocked FOXM1 and EGFR-ERBB2 signaling by either genetically or pharmacologically, then evaluated the effects of thiostrepton and lapatinib on OC proliferation, cell cycle changes, and migratory effects by trypan blue exclusion, 3-D spheroid formation ability, growth assays, confocal microscopy, cell cycle, apoptosis analysis, and in vivo tumor growth assay. Results: In our study, we identified the mechanism of how tumor cells relieve their dependency on EGFR-ERBB2 signaling and adopt alternative and sustainable signaling mechanisms like FOXM1 for resistance to ERBB-therapy and metastasis. To characterize the mechanism of lapatinib and thiostrepton resistance in OC cells, we developed lapatinib and thiostrepton resistant OC cells. We observed that long-term treatment of lapatinib decreased, the levels of pEGFR (Y1068) and pERBB2 (Y1248) proteins, whereas the levels of both phosphorylated and total protein of CDK1 and pPLK1 were upregulated. Strikingly, we found that FOXM1 level is upregulated in the cells are resistant to lapatinib. Next, we determined the nuclear level of FOXM1 which is important for the transcriptional activation of oncogenes. Importantly, we found an increased localization of FOXM1 in the nucleus in lapatinib resistant cells. In contrast, we found that the thiostrepton resistant OC cells express high levels of EGFR and ERBB2. In conjunction, we found that combined FOXM1 and EGFR-ERBB2 inhibition by the sub-lethal concentration of thiostrepton and lapatinib treatment reduced EGFR-ERBB2 and FOXM1 expression and induced apoptosis and inhibition of the colony-forming ability of tumor cells more than a single agent treatment. In addition, our Western blots, immunofluorescence, and immunohistochemistry assays in combination with in vivo studies demonstrated that the combined pharmacological inhibition of FOXM1 by thiostrepton reduced nuclear expression of the FOXM1 in lapatinib resistant cells while EGFR-ERBB2 inhibitor lapatinib downregulates the EGFR-ERBB2 in thiostrepton resistant cells. Taken together, our data demonstrated that the lapatinib resistant mechanism operates through FOXM1 activation likely through CDK1 or PLK1. To inhibit the levels of FOXM1, we used thiostrepton, which is capable of inhibiting the levels of FOXM1 in OC cells, and the cells which are resistant to thiostrepton express high levels of p-EGFR, p-ERBB2, and p-ERK2. In support to our results, we found that thiostrepton resistant cells express high levels of pEGFR and pERBB2, as well as high levels of EGF mRNA. Conclusions: Taken together, our data demonstrated that FOXM1 and EGFR-ERBB2 expression and activation provide a compensatory mechanism against EGFR/ERBB2 or FOXM1 therapy. Thus, inhibiting FOXM1 and EGFR/ERBB2 levels would provide an opportunity to sensitize OC cells resistant to FOXM1 or EGFR/ERBB2 therapy in OC patients.

Synergistic Activity of the HSP90 Inhibitor Ganetespib With Lapatinib Reverses Acquired Lapatinib Resistance in HER2-Positive Breast Cancer Cells.

Lapatinib is an FDA-approved EGFR and HER2 tyrosine kinase inhibitor for the treatment of HER2-positive breast cancer patients. However, its therapeutic efficacy is limited by primary or acquired resistance. In the present study, we established breast cancers cells with acquired lapatinib resistance and investigated the antitumor activity of the second-generation HSP90 inhibitor ganetespib in association with lapatinib in lapatinib-sensitive and -resistant cells. The combination treatment showed synergistic inhibition of HER and the downstream PI3K/Akt and Ras/MEK/ERK pathways, in addition to enhancing induction of early apoptotic cell death and G1 arrest in both parent and lapatinib-resistant cells in vitro. The joint administration of ganetespib and lapatinib depleted the aberrant nuclear transcription factor STAT3, a mediator of the cell cycle and apoptosis-related pathways that is probably involved in the lapatinib resistance of HER2-positive breast cancer cells. In conjunctive with the augmented inhibition of tumor growth observed in both SKBR3 and SKBR3-L xenografts compared to monotherapy, our data provide a sound preclinical basis for combination treatment with lapatinib and ganetespib for refractory HER2-positive breast cancer.

Abstract LB132: CDK8/19 inhibition overcomes in vitro and in vivo resistance to lapatinib in HER2+ breast cancer via STAT1 and STAT3

Abstract One of every five breast cancers are driven by gene amplification and overexpression of HER2/ERBB2 tyrosine kinase. HER2+ breast cancers are treated both by monoclonal antibodies (such as trastuzumab) and small-molecule HER2 kinase inhibitors (such as lapatinib). However, some HER2+ tumors are inherently resistant to HER2-targeting drugs and most of metastatic cancers treated with such drugs eventually acquire resistance. We have analyzed the effect of small-molecule inhibitors of transcriptional serine/threonine kinases CDK8 and CDK19, paralogs of the Mediator kinase that modulates the activity of several signal-responsive transcription factors, on the response to lapatinib in a panel of HER2+ breast cancer cell lines. Selective CDK8/19 inhibitors senexin B and SNX631 showed in vitro synergy with lapatinib in different lapatinib-responsive cell lines and overcame lapatinib resistance in cells with either inherent or acquired resistance to this drug. In vivo treatment with SNX631 significantly inhibited the growth of cell-line based and a patient-derived xenograft (PDX) models of HER2+ breast cancer. A combination of SNX631 and lapatinib drastically suppressed the growth of tumors formed by lapatinib-sensitive HCC1954 cells and their lapatinib-resistant derivative HCC1954-L, overcoming lapatinib resistance of the latter. GSEA analysis of RNA-Seq data from cell treated in vitro with lapatinib, senexin B or their combination, showed that lapatinib activated the JAK/STAT pathway but the addition of the CDK8/19 inhibitor suppressed such induction. CDK8/19 inhibitors, when combined with lapatinib, suppressed STAT3 and STAT1 phosphorylation at Ser727. CRISPR/CAS9 knockout of STAT1 and STAT3 together (but not individually) sensitized HER2+ breast cancer cells to lapatinib and reduced the lapatinib-sensitizing effect of CDK8/19 inhibitors, suggesting that STAT1 and STAT3 mediate the effect of CDK8/19. Through miRNA-Seq and QPCR analysis, we have also identified two miRNAs that are regulated by STAT1 or STAT3 and overexpressed in lapatinib-resistant cells. These miRNAs are upregulated upon lapatinib treatment, whereas the combination of lapatinib with senexin B decreases their expression, suggesting that these miRNAs may mediate the effects of CDK8/19 and STAT1/3 on lapatinib resistance. Our results suggest that combining HER2- and CDK8/19-targeting drugs could be a promising therapeutic direction for HER2+ breast cancers after the failure of HER2-targeted therapy. Citation Format: Xiaokai Ding, Amanda C. Sharko, Martina S-J McDermott, Hao Ji, Alexander Chumanevich, Gary P. Schools, Zachary T. Mack, Elena Pugacheva, Victor Tatarskiy, Anastasia Khrustaleva, Alexander Tyakht, Michael Shtutman, Mengqian Chen, Igor Roninson, Eugenia V. Broude. CDK8/19 inhibition overcomes in vitro and in vivo resistance to lapatinib in HER2+ breast cancer via STAT1 and STAT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB132.

Abstract 2453: Resistance of triple-negative breast cancer cells to EGFR-targeted tyrosine kinase inhibitors is associated with Annexin A6-dependent metabolic reprogramming

Abstract The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in multiple tumor types, including triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) remains a hard-to-treat breast cancer subtype. Significant effort has thus far been devoted to epidermal growth factor receptor (EGFR) targeted therapies as 70% of TNBC tumors overexpress EGFR. However, clinical trials utilizing anti-EGFR therapies have shown great success in various cancer types including non-small cell lung cancer but have been largely unsuccessful in TNBC due to the lack of understanding of TKI-acquired resistance. We have shown that the expression status of annexin-A6 (AnxA6), a calcium-dependent membrane binding tumor suppressor, stabilizes activated EGFR on the cell surface. We have also shown that chronic treatment of AnxA6-low TNBC cells with Lapatinib induces AnxA6 expression and accumulation of cholesterol in late endosomes suggesting a novel mechanism for resistance of TNBC to EGFR-TKIs. Other studies have revealed that AnxA6 expression contributes to impaired fat storage and adiponectin release in adipose tissues, and that lack of AnxA6 compromises alanine-dependent gluconeogenesis and liver regeneration in mice. However, whether the EGFR-TKI induced upregulation of AnxA6 is associated with metabolic reprogramming in TNBC cells remains unclear. Using the Seahorse XF Analyzer, we assessed the live-cell oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TNBC cells. We demonstrate that the basal mitochondrial respiration and glycolytic rates classified TNBC cells into two distinct metabolic phenotypes. Those with higher OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 high cells (energetic phenotype), while those with lower OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 low cells (quiescentphenotype). Induction of AnxA6 expression following chronic treatment of AnxA6-low TNBC cells with Lapatinib (LapR) reverted these otherwise quiescent metabolic phenotypes to the energetic phenotype associated with higher OCR/ECAR ratios and cellular ATP production capacity. However, downregulation of AnxA6 in AnxA6 high TNBC cells attenuated the mitochondrial and glycolytic function with reduced OCR/ECAR ratios and decreased cellular ATP production capacity. NMR-based metabolomics revealed that AnxA6 depleted and/or lapatinib-resistant TNBC cells have a greater dependency on gluconeogenic amino acids including alanine, cysteine, glycine, and proline. These data suggest that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and provide novel insights into the failure of EGFR-targeted therapies as therapeutic options for TNBC. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Resistance of triple-negative breast cancer cells to EGFR-targeted tyrosine kinase inhibitors is associated with Annexin A6-dependent metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2453.

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

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

The Role of Annexin‐A6 in the Metabolic Reprogramming and EGFR‐TKI resistance in Triple‐negative breast cancer cells

The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying therapeutic resistance in multiple tumor types, including triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) remains a hard-to-treat breast cancer subtype. Significant effort has thus far been devoted to epidermal growth factor receptor (EGFR) targeted therapies as 70% of TNBC tumors overexpress EGFR. However, clinical trials utilizing anti-EGFR therapies have shown great success in various cancer types including non-small cell lung cancer but have been largely unsuccessful in TNBC due to the lack of understanding of TKI-acquired resistance. We have shown that the expression status of annexin-A6 (AnxA6), a calcium-dependent membrane binding tumor suppressor, stabilizes activated EGFR on the cell surface. We have also shown that chronic treatment of AnxA6-low TNBC cells with Lapatinib induces AnxA6 expression and accumulation of cholesterol in late endosomes suggesting a novel mechanism for resistance of TNBC to EGFR-TKIs. Other studies have revealed that AnxA6 expression contributes to impaired fat storage and adiponectin release in adipose tissues, and that lack of AnxA6 compromises alanine-dependent gluconeogenesis and liver regeneration in mice. However, whether the EGFR-TKI induced upregulation of AnxA6 is associated with metabolic reprogramming in TNBC cells remains unclear. Using the Seahorse XF Analyzer, we assessed the live-cell oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of TNBC cell lines. We demonstrate that the basal mitochondrial respiration and glycolytic rates classified TNBC cell lines into two distinct metabolic phenotypes. Those with higher OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 high cells (energetic phenotype), while those with lower OCR/ECAR ratios and cellular ATP production capacity were mostly AnxA6 low cells (quiescent phenotype). Induction of AnxA6 expression following chronic treatment of AnxA6-low TNBC cells with Lapatinib (LapR) reverted these otherwise quiescent metabolic phenotypes to the energetic phenotype associated with higher OCR/ECAR ratios and cellular ATP production capacity. However, down regulation of AnxA6 in AnxA6 high TNBC cells attenuated the mitochondrial and glycolytic function with reduced OCR/ECAR ratios and decreased cellular ATP production capacity. NMR-based metabolomics revealed that AnxA6 depleted and/or lapatinib-resistant TNBC cells have a greater dependency on gluconeogenic amino acids including alanine, cysteine, glycine, and proline. These data suggest that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and provide novel insights into the failure of EGFR-targeted therapies as therapeutic options for TNBC.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer

BackgroundAround 15–20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance.MethodsRANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling.ResultsRANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status.ConclusionsOur data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Abstract PS16-28: Runx3 regulates emt during development lapatinib resistance

Abstract RUNX3 regulates EMT during development lapatinib resistanceBackground: Lapatinib is a small molecular inhibitor of HER2 and EGFR tyrosine kinase, which is used for HER2 positive metastatic breast cancer patients. However, significant proportion of patients relapse due to acquired resistance. The epithelial to mesenchymal transition (EMT), which induces cell migration, invasion and cancer stem cell progression, was one of the mechanism that confers to drug resistance. We determined the correlation of lapatinib resistance and EMT, and identified a regulatory factor of EMT which associated with the development process of acquired lapatinib resistance.Methods: Acquired lapatinib resistant SK-BR-3 (SK-BR-3 LR) cells were established by continuously exposing to lapatinib for 7 months. The sensitivity of lapatinib was confirmed by MTT assay. Cell cycle progression was verified using flow cytometry analysis. Expression of signal transduction molecules were determined using quantitative PCR, western blotting and transcriptome data analysis. Cell migration and invasion ability were verified using wound healing assay and Boyden chamber assay. siRNA knock-down system were used for further analysis. Results: Lapatinib resistant (SK-BR-3 LR) cells showed aggressive morphology compared with parental cells. The growth rate and cell cycle progression were increased in SK-BR-3 LR cells. Expression of Snail, Vimentin, SOX2, Nanog, TGF-b1 and Smad proteins increased in SK-BR-3 LR cells. In SK-BR-3 LR cells, cell migration and invasion were significantly increased. Correlated with a promoter methylation of RUNX3, expression of RUNX3 was down-regulated in SK-BR-3 LR cells. In Runx3 knock-down cells TGF-b1, SOX2, smad molecules were up-regulated. Conclusion: SK-BR-3 LR cells showed aggressive phenotype. Expression of EMT markers, cancer stem cell markers, TGF-β pathway associated molecules and Smad proteins were increased in SK-BR-3 LR cells. Also, the increase of cell migration and invasion was observed in LR cells. RUNX3 affected to cell migration and invasion through regulation of EMT associated molecules. Therefore, RUNX3 might be a specified molecule, which partially contributes resistance to lapatinib through regulates EMT. Citation Format: So Hyeon Kim, Ahrum Min, Seongyeong Kim, Yoonjung Park, Yu Jin Kim, Seock-Ah Im. Runx3 regulates emt during development lapatinib resistance [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-28.

HSP90 Inhibitor, 17-DMAG, Alone and in Combination with Lapatinib Attenuates Acquired Lapatinib-Resistance in ER-positive, HER2-Overexpressing Breast Cancer Cell Line.

Simple SummaryLapatinib is a tyrosine kinase inhibitor widely used as a treatment for a Human Epidermal growth factor Receptor 2 (HER2) (+) breast cancer patients. However, when resistance is acquired through continued exposure, and it is associated with a poor prognosis for patients. In this study, we identified HSP90 as a common node for acquired resistance to lapatinib in two lapatinib resistant cell lines using proteomic analysis. Notably, in vitro and in vivo studies demonstrated synergy effect between lapatinib and an HSP90 inhibitor were observed in the estrogen receptor (+) HER2 (+) breast cancer cell only. These results could be a potential strategy for future clinical trials for HSP90 inhibitors in treatment—refractory HER2 (+) metastatic cancer patientsLapatinib, a Human Epidermal growth factor Receptor 2 (HER2)-targeting therapy in HER2-overexpressing breast cancer, has been widely used clinically, but the prognosis is still poor because most patients acquire resistance. Therefore, we investigated mechanisms related to lapatinib resistance to evaluate new therapeutic targets that may overcome resistance. Lapatinib-resistant cell lines were established using SKBR3 and BT474 cells. We evaluated cell viability and cell signal changes, gene expression and protein changes. In the xenograft mouse model, anti-tumor effects were evaluated using drugs. Analysis of the protein interaction network in two resistant cell lines with different lapatinib resistance mechanisms showed that HSP90 protein was commonly increased. When Heat Shock Protein 90 (HSP90) inhibitors were administered alone to both resistant cell lines, cell proliferation and protein expression were effectively inhibited. However, inhibition of cell proliferation and protein expression with a combination of lapatinib and HSP90 inhibitors showed a more synergistic effect in the LR-BT474 cell line than the LR-SKBR3 cell line, and the same result was exhibited with the xenograft model. These results suggest that HSP90 inhibitors in patients with lapatinib-resistant Estrogen Receptor (ER) (+) HER2 (+) breast cancer are promising therapeutics for future clinical trials.

Abstract 1916: Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma

Abstract Background: A major target for esophageal adenocarcinoma (EAC) therapies is the human epidermal growth factor receptor 2 (HER2). EAC overexpresses HER2 and lapatinib, a dual tyrosine kinase inhibitor blocking HER1 and HER2 pathways fails to improve patient survival. Molecular mechanisms of this lapatinib resistance are still unclear. We therefore explored the role of glycolytic enzyme pyruvate kinase M2 (PKM2), a key regulator of Warburg effect in lapatinib resistance mechanism of EAC cells. Methods: First we established lapatinib-resistant OE19 (LPR-OE19) cells from OE19 EAC cells and characterized it. We then investigated the comparative cell growth inhibition, apoptotic and lactate production effects of HER2 inhibitor lapatinib and PKM2 inhibitor shikonin either alone or in combinations, both in OE19 and LPR-OE19 cells with and without knockdown of PKM2 and HSP40 by siRNAs. We then explored the antitumor efficacy following lapatinib and shikonin mono and combination therapies in murine subcutaneous xenograft model of human EAC. Results: Lapatinib-resistant OE19 (LPR-OE19) cells were established from OE19 cells by intermittent exposure to increasing concentrations of lapatinib for a period of total six months. Lapatinib resistant LPR-OE19 cells showed downregulation of HSP40 both at protein and mRNA levels whereas showed upregulation of PKM2 only at protein level. LPR-OE19 cells showed significantly reduced sensitivity to lapatinib induced cell growth inhibition, apoptosis and decreased cell migration compared to parent OE19 cells. Interestingly, augmented cell growth inhibition, apoptosis and decreased cell migration were observed in LPR-OE19 cells compared to parent OE19 cells when lapatinib was combined with shikonin. More interestingly, knockdown of PKM2 in LPR-OE19 cells abolished the reduced sensitivity of lapatinib induced cell growth inhibition and also abolished augmented cell growth inhibition response when lapatinib and shikonin were combined. Contrary to that, knockdown of HSP40 in OE19 cells enhanced PKM2 expression leading to significantly reduced sensitivity to lapatinib induced growth inhibition with augmented growth inhibition when lapatinib and shikonin were combined. Moreover, LPR-OE19 cells showed enhanced lactate production compared to parent OE19. Interestingly, PKM2 and HSP40 co-localize with each other in the cell nucleus suggesting that PKM2 bound to molecular chaperone HSP40. In subcutaneous xenografts using LPR-OE19 cells, average net tumor growth after two weeks in different therapy groups was 494.72 mm3 in control, 403.11 mm3 after lapatinib (p=0.241), 339.48 mm3 after shikonin (p=0.0478), and 141.59 mm3 after shikonin plus lapatinib (p=0.0003). Conclusions: In conclusion, these data suggest that combination therapy with lapatinib and glycolytic inhibitor shikonin should be tested as a potent option for lapatinib resistant HER2-positive EAC patients and could be a novel treatment strategy for specific EAC. In addition, it is suggested that targeting HSP40-PKM2 interaction as an innovative therapeutic approach to overcome lapatinib resistance in EAC. Citation Format: Md Sazzad Hassan, Samantha Holmes, Victoria Makuru, Chloe Johnson, Urs von Holzen. Targeting Warburg effect to overcome lapatinib resistance in esophageal adenocarcinoma [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 1916.