Martina McDermott

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance.

PP2A inhibition overcomes acquired resistance to HER2 targeted therapy

BackgroundHER2 targeted therapies including trastuzumab and more recently lapatinib have significantly improved the prognosis for HER2 positive breast cancer patients. However, resistance to these agents is a significant clinical problem. Although several mechanisms have been proposed for resistance to trastuzumab, the mechanisms of lapatinib resistance remain largely unknown. In this study we generated new models of acquired resistance to HER2 targeted therapy and investigated mechanisms of resistance using phospho-proteomic profiling.ResultsLong-term continuous exposure of SKBR3 cells to low dose lapatinib established a cell line, SKBR3-L, which is resistant to both lapatinib and trastuzumab. Phospho-proteomic profiling and immunoblotting revealed significant alterations in phospho-proteins involved in key signaling pathways and molecular events. In particular, phosphorylation of eukaryotic elongation factor 2 (eEF2), which inactivates eEF2, was significantly decreased in SKBR3-L cells compared to the parental SKBR3 cells. SKBR3-L cells exhibited significantly increased activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF2. SKBR3-L cells showed increased sensitivity to PP2A inhibition, with okadaic acid, compared to SKBR3 cells. PP2A inhibition significantly enhanced response to lapatinib in both the SKBR3 and SKBR3-L cells. Furthermore, treatment of SKBR3 parental cells with the PP2A activator, FTY720, decreased sensitivity to lapatinib. The alteration in eEF2 phosphorylation, PP2A activity and sensitivity to okadaic acid were also observed in a second HER2 positive cell line model of acquired lapatinib resistance, HCC1954-L.ConclusionsOur data suggests that decreased eEF2 phosphorylation, mediated by increased PP2A activity, contributes to resistance to HER2 inhibition and may provide novel targets for therapeutic intervention in HER2 positive breast cancer which is resistant to HER2 targeted therapies.

Neuromedin U: A Candidate Biomarker and Therapeutic Target to Predict and Overcome Resistance to HER-Tyrosine Kinase Inhibitors

Intrinsic and acquired resistance to HER-targeting drugs occurs in a significant proportion of HER2-overexpressing breast cancers. Thus, there remains a need to identify predictive biomarkers that could improve patient selection and circumvent these types of drug resistance. Here, we report the identification of neuromedin U (NmU) as an extracellular biomarker in cells resistant to HER-targeted drugs. NmU overexpression occurred in cells with acquired or innate resistance to lapatinib, trastuzumab, neratinib, and afatinib, all of which displayed a similar trend upon short-term exposure, suggesting NmU induction may be an early response. An analysis of 3,489 cases of breast cancer showed NmU to be associated with poor patient outcome, particularly those with HER2-overexpressing tumors independent of established prognostic indicators. Ectopic overexpression of NmU in drug-sensitive cells conferred resistance to all HER-targeting drugs, whereas RNAi-mediated attenuation sensitized cells exhibiting acquired or innate drug resistance. Mechanistic investigations suggested that NmU acted through HSP27 as partner protein to stabilize HER2 protein levels. We also obtained evidence of functional NmU receptors on HER2-overexpressing cells, with the addition of exogenous NmU eliciting an elevation in HER2 and EGFR expression along with drug resistance. Finally, we found that NmU seemed to function in cell motility, invasion, and anoikis resistance. In vivo studies revealed that NmU attenuation impaired tumor growth and metastasis. Taken together, our results defined NmU as a candidate drug response biomarker for HER2-overexpressing cancers and as a candidate therapeutic target to limit metastatic progression and improve the efficacy of HER-targeted drugs.

Dual inhibition of IGF1R and ER enhances response to trastuzumab in HER2 positive breast cancer cells

Although HER2 targeted therapies have improved prognosis for HER2 positive breast cancer, HER2 positive cancers which co-express ER have poorer response rates to standard HER2 targeted therapies, combined with chemotherapy, than HER2 positive/ER negative breast cancer. Administration of hormone therapy concurrently with chemotherapy and HER2 targeted therapy is generally not recommended. Using publically available gene expression datasets we found that high expression of IGF1R is associated with shorter disease-free survival in patients whose tumors are ER positive and HER2 positive. IGF1R is frequently expressed in HER2 positive breast cancer and there is significant evidence for crosstalk between IGF1R and both HER2 and ER. Therefore, we evaluated the therapeutic potential of targeting ER and IGF1R in cell line models of HER2/ER/IGF1R positive breast cancer, using tamoxifen and two IGF1R targeted tyrosine kinase inhibitors (NVP-AEW541 and BMS-536924). Dual inhibition of ER and IGF1R enhanced growth inhibition in the four HER2 positive cell lines tested and caused an increase in cell cycle arrest in G1 in BT474 cells. In addition, combined treatment with trastuzumab, tamoxifen and either of the IGF1R TKIs enhanced response compared to dual targeting strategies in three of the four HER2 positive breast cancer cell lines tested. Furthermore, in a cell line model of trastuzumab-resistant HER2 positive breast cancer (BT474/Tr), tamoxifen combined with an IGF1R TKI produced a similar enhanced response as observed in the parental BT474 cells suggesting that this combination may overcome acquired trastuzumab resistance in this model. Combining ER and IGF1R targeting with HER2 targeted therapies may be an alternative to HER2 targeted therapy and chemotherapy for patients with HER2/ER/IGF1R positive breast cancer.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL

BackgroundLapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells.MethodsWe examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer.ResultsIn this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells.ConclusionsOur findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.

Abstract 1512: Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor

The Mediator complex-associated cyclin-dependent kinase CDK8 is an oncogenic transcription-regulating serine/threonine kinase that mediates multiple cancer-associated transcriptional pathways. Despite recent high-profile attention to CDK8 as a novel cancer drug target, very little is known about the function of CDK8’s closely related paralog CDK19. Using CRISPR/Cas9n system we generated CDK8/CDK19 single-knockout (CDK8-KO and CDK19-KO) and double-knockout (CDK8/19-dKO) derivatives of HEK293 cells. RNA-Seq was used to characterize the effects of a highly selective small-molecule CDK8/19 kinase inhibitor Senexin B on gene expression in the parental, single-knockout and double-knockout cell lines. This analysis was conducted in the absence or in the presence of TNF-alpha, an inducer of transcription factor NF-kappa-B that we have previously shown to be potentiated by CDK8. The following results were obtained. (1) CDK8 and CDK19 have complementary functions in stabilization of their partner Cyclin C (independent of their kinase activity) and phosphorylation of transcription factor STAT1 at S727 (dependent on the kinase activity). (2) Senexin B treatment affected gene expression in wild-type 293 cells but had almost no effect in CDK8/19-dKO cells, indicating a very high degree of target selectivity. (3) In contrast to the results with CDK8/19-dKO, most of the genes affected by Senexin B in the wild-type cells were also affected in CDK8-KO and CDK19-KO cells, indicating complementary functions of CDK8 and CDK19. (4) Re-expression of either CDK8 or CDK19, but not of a CDK8 kinase-dead (D173A) mutant, in CDK8/19-dKO cells restored CDK8/19 kinase-dependent gene expression as well as the regulatory effects of Senexin B. (5) Many more genes were inhibited rather than induced by Senexin B, indicating that CDK8/19 act primarily as positive regulators of transcription. (6) The total number of genes affected by Senexin B was greatly increased in cells treated with TNF-alpha, suggesting that cooperation with other transcription factors (such as NF-kappa-B) is the primary role of CDK8/19. Our results indicate that complete suppression of cancer-relevant activities of CDK8 requires simultaneous inhibition of both CDK8 and CDK19. Citation Format: Mengqian Chen, Bing Hu, Hao Ji, Serena Altilia, Jiaxin Liang, Martina McDermott, Chang-uk Lim, Donald C. Porter, Eugenia Broude, Igor Roninson. Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2017-1512

P5-06-09: Acquired Sensitivity to TRAIL Mediated Apoptosis in Lapatinib Resistant SKBR3 Cells.

Introduction Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor is approved for the treatment of trastuzumab-refractory HER2 positive metastatic breast cancer. However, not all HER2 positive tumors respond to lapatinib and patients who initially respond frequently relapse. Understanding the molecular alterations associated with acquisition of lapatinib resistance may lead to the identification of targets to overcome resistance. Thus, the aim of this study was to examine changes in apoptosis in a model of acquired lapatinib resistance. Methods SKBR3 cells were treated with 250 nM lapatinib twice weekly for 6 months to establish the lapatinib resistant cell line SKBR3-L. The effects of TNF-related apoptosis-inducing ligand (TRAIL) and tumour necrosis factor-α (TNF-α) on cell survival and apoptosis induction were examined in SKBR3 and SKBR3-L cells, using the acid phosphatase proliferation assay and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Cleavage of poly ADP ribose polymerase (PARP) was used to confirm apoptosis. TRAIL-1 and TRAIL-2 receptor expression were measured by flow cytometry (FACS). Results In SKBR3-L cells, TRAIL treatment (25 ng/ml) significantly inhibited cell survival (94.0 ± 12.0 %) compared to the SKBR3 cells (11.0 ± 2.2%). Treatment with TNF-α (0.5 ng/ml) also resulted in significant inhibition of cell survival (68.2 ± 10.1 %) in SKBR3-L cells compared to SKBR3 cells (9.4 ± 9.0 %). TRAIL (25 ng/ml) induced a low level of apoptosis in SKBR3 cells (7.0 ± 4.0%), whereas in SKBR3-L cells, TRAIL (25 ng/ml) induced significant apoptosis (54.8 ± 2.6%, p Conclusions SKBR3-L cells, a model of acquired lapatinib resistance, display significant sensitivity to TRAIL induced apoptosis compared to the parental cell line SKBR3. Thus, targeting TRAIL may represent a novel therapeutic strategy to overcome acquired lapatinib resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-09.

P1-12-06: The Role of MAPK and PI3K/AKT/mTOR Signaling in Innate Lapatinib Resistance.

Background: Lapatinib is a tyrosine kinase inhibitor which blocks downstream MAPK and P13K/AKT/mTOR proliferation and survival signaling pathways in HER2 positive breast cancer cell lines, tumor xenografts and HER2 positive breast cancer patients. However, pre-clinical and clinical studies have shown that not all HER2 positive patients respond to lapatinib and thus innate resistance to lapatinib exists. The identification of biomarkers of lapatinib response is therefore critical and would enable individual therapeutic decisions to be based on tumor biology rather than basic histopathology data alone. The aim of this study was to examine the role of MAPK and PI3K/AKT/mTOR signaling in a panel of lapatinib sensitive and resistant HER2−amplified breast cancer cell lines to identify pharmacodynamic markers of response to lapatinib treatment. Methods: Dose response curves were generated to determine sensitivity to lapatinib in a panel of 17 HER2−amplified breast cancer cell lines. Total and phosphorylated levels of HER2, HER3, EGFR, AKT, ERK, S6K and eEF2 were determined following 24 hours lapatinib treatment in each of the cell lines. Results: Twelve of the cell lines were sensitive to lapatinib with IC50g 1 μM. Levels of pHER2 and pHER3 were decreased in response to lapatinib in all cell lines independent of sensitivity to lapatinib. This suggests that inhibition of HER2/HER3 activation is not indicative of response to lapatinib treatment. There was also no correlation between the levels of HER2, HER3, and EGFR and sensitivity to lapatinib in the cell line panel. In lapatinib sensitive cell lines, lapatinib decreased PI3K (pAKT), mTOR (pS6K) and MAPK (pERK) signaling and increased peEF2 levels. In contrast the levels of pAKT, pS6K, peEF2 and pERK were maintained following lapatinib treatment in lapatinib resistant cells. The continued activation of these proteins in the presence of lapatinib suggests a possible feedback mechanism that warrants further investigation. These data confirm that maintained signaling through either the P13K/AKT/mTOR pathway or the MAPK pathways in the presence of lapatinib can be an early pharmacodynamic biomarker of response. Conclusions: Maintenance of pAKT, pS6K, peEF2 and pERK levels, in response to lapatinib treatment correlates with lapatinib resistance. These data suggest that alterations in the P13K/AKT/mTOR and MAPK pathways play an important role in innate lapatinib resistance and pharmacologically targeting these pathways is a rational therapeutic approach for overcoming innate lapatinib resistance. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-06.

Abstract 3633: eEF2 in acquired lapatinib resistance in HER2 positive breast cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Approximately 25% of breast cancers overexpress HER2. Although HER2 targeted therapies, trastuzumab and more recently lapatinib, have improved prognosis for HER2 breast cancer patients not all HER2 positive tumors respond to HER2 targeted therapies. Using phosphoproteomic profiling, we examined mechanisms of resistance in an in vitro model of acquired lapatinib resistance. SKBR3 cells were conditioned in lapatinib containing media (200 nM) for six months, after which time the resulting SKBR3-L cells exhibited significantly reduced growth inhibition (27.7 ± 0.1 %) when treated with lapatinib (1 µM) treatment compared to parental SKBR3 (90.7 ± 0.2 %) (p<0.01). Using phosphoproteomic profiling, eukaryotic elongation factor 2 (eEF2) was identified as a phosphoprotein which was significantly altered in the SKBR3-L cells compared to the parental SKBR3 cells. One form of phospho-eEF2 was up-regulated and 6 forms were down-regulated in SKBR3-L compared to SKBR3. eEF2 is a phosphoprotein with an essential role in protein synthesis. Phosphorylation of eEF2 at Thr56 and Thr58 by eEF2 kinase inhibits eEF2 activity. eEF2 kinase activity is regulated by the mTOR pathway, via phosphorylation by p70S6 kinase. To examine the role of eEF2 in lapatinib resistance, levels of phospho-eEF2, eEF2, phospho-p70S6K and p70S6K, were examined in a panel of HER2 positive breast cancer cell lines following 24 hour treatment with lapatinib (1 µM). Treatment with lapatinib resulted in a decrease in phospho-p70S6K and an increase in phospho-eEF2, with no change in the levels of total protein, in lapatinib sensitive cell lines. In order to further examine the role the mTOR pathway and eEF2, plays in acquired resistance to lapatinib, the effect of rapamycin, alone and in combination with lapatinib, was investigated in SKBR3 par and SKBR3-L cells. Combined treatment with lapatinib (200 nM) and rapamycin (2 nM) results in similar inhibition of growth (64.5 ± 3.3 %) in SKBR3-L cells as lapatinib alone in SKBR3 parental cells (62.5 ± 0.6 %). In conclusion, response to lapatinib is associated with decreased eEF2 activity in HER2 positive cell lines. Furthermore, inhibition of mTOR signaling, which causes inhibition of eEF2 activity, appears to restore sensitivity to lapatinib in our model of acquired lapatinib resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3633.