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

ABSTRACT: 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.

SUBMITTER: Ye M

PROVIDER: S-EPMC8287059 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The MEK/MAPK pathway is commonly activated in HER2-positive breast cancer, but little investigation of targeting this pathway has been undertaken. Here we present the results of an in vitro preclinical evaluation of refametinib, an allosteric MEK1/2 inhibitor, in HER2-positive breast cancer cell lines including models of acquired resistance to trastuzumab or lapatinib.A panel of HER2-positive breast cancer cells were profiled for mutational status and also for anti-proliferative response to refametinib alone and in combination with the PI3K inhibitor (PI3Ki) copanlisib and the HER2-targeted therapies trastuzumab and lapatinib. Reverse phase protein array (RPPA) was used to determine the effect of refametinib alone and in combination with PI3Ki and HER2-inhibitors on expression and phosphorylation of proteins in the PI3K/AKT and MEK/MAPK pathways. We validated our proteomic in vitro findings by utilising RPPA analysis of patients who received either trastuzumab, lapatinib or the combination of both drugs in the NCT00524303/LPT109096 clinical trial.Refametinib has anti-proliferative effects when used alone in 2/3 parental HER2-positive breast cancer cell lines (HCC1954, BT474), along with 3 models of these 2 cell lines with acquired trastuzumab or lapatinib resistance (6 cell lines tested). Refametinib treatment led to complete inhibition of MAPK signalling. In HCC1954, the most refametinib-sensitive cell line (IC50= 397 nM), lapatinib treatment inhibits phosphorylation of MEK and MAPK but activates AKT phosphorylation, in contrast to the other 2 parental cell lines tested (BT474-P, SKBR3-P), suggesting that HER2 may directly activate MEK/MAPK and not PI3K/AKT in HCC1954 cells but not in the other 2 cell lines, perhaps explaining the refametinib-sensitivity of this cell line. Using RPPA data from patients who received either trastuzumab, lapatinib or the combination of both drugs together with chemotherapy in the NCT00524303 clinical trial, we found that 18% (n=38) of tumours had decreased MAPK and increased AKT phosphorylation 14 days after treatment with HER2-targeted therapies. The combination of MEK inhibition (MEKi) with refametinib and copanlisib led to synergistic inhibition of growth in 4/6 cell lines tested (CI @ED75 = 0.39-0.75), whilst the combinations of lapatinib and refametinib led to synergistic inhibition of growth in 3/6 cell lines (CI @ED75 = 0.39-0.80).Refametinib alone or in combination with copanlisib or lapatinib could represent an improved treatment strategy for some patients with HER2-positive breast cancer, and should be considered for clinical trial evaluation. The direct down-regulation of MEK/MAPK but not AKT signalling by HER2 inhibition (e.g. by lapatinib or trastuzumab), which we demonstrate occurs in 18% of HER2-positive breast cancers may serve as a potential biomarker of responsiveness to the MEK inhibitor refametinib.

Project description:BACKGROUND:Lapatinib 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. METHODS:We 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. RESULTS:In 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. CONCLUSIONS:Our 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.

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

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