PI3K inhibition enhances the anti-tumor effect of eribulin in triple negative breast cancer.
ABSTRACT: Loss of the tumor suppressor phosphatase and tensin homolog (PTEN) is commonly observed in triple negative breast cancer (TNBC), leading to activation of the phosphoinositide 3-kinase (PI3K) signaling to promote tumor cell growth and chemotherapy resistance. In this study, we investigated whether adding a pan-PI3K inhibitor could improve the cytotoxic effect of eribulin, a non-taxane microtubule inhibitor, in TNBC patient-derived xenograft models (PDX) with loss of PTEN, and the underlying molecular mechanisms. Three TNBC-PDX models (WHIM6, WHIM12 and WHIM21), all with loss of PTEN expression, were tested for their response to BKM120 and eribulin, alone or in combination in vivo. In addition, the effect of drug treatment on cell proliferation and cell cycle progression were also performed in vitro using a panel of TNBC cell lines, including 2 derived from PDX models. The combination of eribulin and BKM120 led to additive or synergistic anti-tumor effect in 2 of the 3 PDX models, accompanied by an enhanced mitotic arrest and apoptosis in sensitive PDX models. In addition, the combination was synergistic in reducing mammosphere formation, and markers for epithelial-mesenchymal transition (EMT). In conclusion, PI3K inhibition induces synergistic anti-tumor effect when combined with eribulin, by enhancing mitotic arrest and apoptosis, as well as, reducing the cancer stem cell population. This study provides a preclinical rationale to investigate the therapeutic potential for the combination of PI3K inhibition and eribulin in the difficult to treat TNBC. Further studies are needed to identify the biomarkers of response for target patient selection.
Project description:Background: Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. Triple negative breast cancers (TNBC) are often PTEN-deficient, making mTOR a compelling target. We evaluated the efficacy of catalytic mTOR inhibitor TAK228 alone and in combination with eribulin in TNBC. Results: Five of eight triple negative breast cell lines were sensitive to TAK228, independent of PIK3CA/PTEN status. Western blotting demonstrated inhibition of mTORC1/2 signaling as demonstrated by decreased phospho-AKT, phospho-S6 and phospho-4EBP1. In vitro, TAK228 was synergistic with eribulin in all eight TNBC cell lines. The combination of TAK228 and eribulin did not enhance apoptosis but increased G2/M growth arrest. In vivo, TAK228 led to modest growth inhibition in TNBC patient-derived xenografts (PDXs) with no tumor regression observed. In two TNBC PDXs with PTEN loss, one with intrinsic eribulin sensitivity, another eribulin resistance, TAK228 in combination with eribulin did not enhance in vivo efficacy. In a third PTEN-negative TNBC model, eribulin alone achieved disease stabilization, but the combination of TAK228 and eribulin led to significantly smaller tumor volumes compared to eribulin alone (p < 0.001). Methods: We tested in vitro efficacy of TAK228 in a panel of TNBC cell lines with cell proliferation assays. In vivo antitumor efficacy of TAK228 was evaluated alone and in combination with eribulin. Conclusion: TAK228 enhances the antitumor efficacy of eribulin in TNBC models in vitro, and enhanced in vivo activity in selected models. Further study is needed to determine the potential of this combination, and optimal patient selection strategies.
Project description:PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.
Project description:Unlike other breast cancer subtypes, patients with triple negative breast cancer (TNBC) have poor outcomes and no effective targeted therapies, leaving an unmet need for therapeutic targets. Efforts to profile these tumors have revealed the PI3K/AKT/mTOR pathway as a potential target. Activation of this pathway also contributes to resistance to anti-cancer agents, including microtubule-targeting agents. Eribulin is one such microtubule-targeting agent that is beneficial in treating taxane and anthracycline refractory breast cancer. In this study, we compared the effect of eribulin on the PI3K/AKT/mTOR pathway with other microtubule-targeting agents in TNBC. We found that the phosphorylation of AKT was suppressed by eribulin, a microtubule depolymerizing agent, but activated by paclitaxel, a microtubule stabilizing agent. The combination of eribulin and everolimus, an mTOR inhibitor, resulted in an increased reduction of p-S6K1 and p-S6, a synergistic inhibition of cell survival in vitro, and an enhanced suppression of tumor growth in two orthotopic mouse models. These findings provide a preclinical foundation for targeting both the microtubule cytoskeleton and the PI3K/AKT/mTOR pathway in the treatment of refractory TNBC.
Project description:Background: PTEN-deficient tumors are dependent on PI3K? activity, making PI3K? a compelling target. We evaluated the efficacy of PI3K? inhibitor AZD8186 on tumors with PTEN loss. Results: In vitro cell viability assay and immunoblotting demonstrated that PTEN loss was significantly correlated with AZD8186 sensitivity in triple negative breast cancer (TNBC) cell lines. Colony formation assay confirmed sensitivity of PTEN-deficient cell lines to AZD8186. AZD8186 inhibited PI3K signaling in PTEN loss TNBC cells. AZD8186 in combination with paclitaxel, eribulin had synergistic effects on growth inhibition in PTEN loss cells. AZD8186 promoted apoptosis in PTEN loss cells which was synergized by paclitaxel. In vivo, AZD8186 had limited activity as a single agent, but enhanced antitumor activity when combined with paclitaxel in MDA-MB-436 and MDA-MB-468 cell-line xenografts. AZD8186 significantly enhanced antitumor efficacy of anti-PD1 antibodies in the PTEN-deficient BP murine melanoma xenograft model, but not in the PTEN-wild-type CT26 xenograft model. Methods: In vitro, cell proliferation and colony formation assays were performed to determine cell sensitivity to AZD8186. Immunoblotting was performed to assess PTEN expression and PI3K signaling activity. FACS was performed to evaluate apoptosis. In vivo, antitumor efficacy of AZD8186 and its combinations were evaluated. Conclusions: AZD8186 has single agent efficacy in PTEN-deficient TNBC cell lines in vitro, but has limited single agent efficacy in vivo. However, AZD8186 has enhanced efficacy when combined with paclitaxel and anti-PD1 in vivo. Further study is needed to determine optimal combination therapies for PTEN-deficient solid tumors.
Project description:Triple-negative breast cancers (TNBC) are characterized by frequent alterations in the PI3K/AKT/mTOR signaling pathway. In this study, we analyzed PI3K pathway activation in 67 patient-derived xenografts (PDX) of breast cancer and investigated the anti-tumor activity of the mTOR inhibitor everolimus in 15 TNBC PDX with different expression and mutational status of PI3K pathway markers. Expression of the tumor suppressors PTEN and INPP4B was lost in 55% and 76% of TNBC PDX, respectively, while mutations in PIK3CA and AKT1 genes were rare. In 7 PDX treatment with everolimus resulted in a tumor growth inhibition higher than 50%, while 8 models were classified as low responder or resistant. Basal-like, LAR (Luminal AR), mesenchymal and HER2-enriched tumors were present in both responder and resistant groups, suggesting that tumor response to everolimus is not restricted to a specific TNBC subtype. Analysis of treated tumors showed a correlation between tumor response and post-treatment phosphorylation of AKT, increased in responder PDX, while PI3K pathway markers at baseline were not sufficient to predict everolimus response. In conclusion, targeting mTOR decreased tumor growth in 7 out of 15 TNBC PDX tested. Response to everolimus occurred in different TNBC subtypes and was associated with post-treatment increase of P-AKT.
Project description:PTEN inactivation occurs commonly in human cancers and putatively activates the PI3K/AKT/ mTOR pathway. Activation of this pathway has been involved in resistance to chemotherapy or anti-EGFR/HER2 therapies. We evaluated the combination of PI3K-mTOR inhibitors with chemotherapy or the pan-HER inhibitor dacomitinib in PTEN-deficient patient-derived tumor xenografts (PDX). Three PDXs were selected for their lack of PTEN expression by immunohistochemistry: a triple-negative breast cancer (TNBC), a KRAS G12R low-grade serous ovarian cancer (LGSOC), and KRAS G12C and TP53 R181P lung adenocarcinoma (LADC). Two dual PI3K-mTOR inhibitors were evaluated-PF-04691502 and PF-05212384-in combination with cisplatin, paclitaxel, or dacomitinib. The addition of PI3K-mTOR inhibitors to cisplatin or paclitaxel increased the activity of chemotherapy in the TNBC and LGSOC models; whereas no added activity was observed in the LADC model. Pharmacodynamic modulation of pS6 and pAKT was observed in the group treated with PI3K-mTOR inhibitor. Our research suggests that the addition of a PI3K-mTOR inhibitor may enhance tumor growth inhibition when compared to chemotherapy alone in certain PTEN-deficient PDXs. However, this benefit was absent in the KRAS and TP53 mutant LADC model. The role of PTEN deficiency in the antitumor activity of these combinations should be further investigated in the clinic.
Project description:Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent.
Project description:BACKGROUND:Selinexor (KPT-330) is an oral agent that has been shown to inhibit the nuclear exporter XPO1. Given the pressing need for novel therapies for triple-negative breast cancer (TNBC), we sought to determine the antitumor effects of selinexor in vitro and in vivo. METHODS:Twenty-six breast cancer cell lines of different breast cancer subtypes were treated with selinexor in vitro. Cell proliferation assays were used to measure the half-maximal inhibitory concentration (IC50) and to test the effects in combination with chemotherapy. In vivo efficacy was tested both as a single agent and in combination therapy in TNBC patient-derived xenografts (PDXs). RESULTS:Selinexor demonstrated growth inhibition in all 14 TNBC cell lines tested; TNBC cell lines were more sensitive to selinexor (median IC50 44 nM, range 11 to 550 nM) than were estrogen receptor (ER)-positive breast cancer cell lines (median IC50?>?1000 nM, range 40 to >1000 nM; P?=?0.017). In multiple TNBC cell lines, selinexor was synergistic with paclitaxel, carboplatin, eribulin, and doxorubicin in vitro. Selinexor as a single agent reduced tumor growth in vivo in four of five different TNBC PDX models, with a median tumor growth inhibition ratio (T/C: treatment/control) of 42% (range 31 to 73%) and demonstrated greater antitumor efficacy in combination with paclitaxel or eribulin (average T/C ratios of 27% and 12%, respectively). CONCLUSIONS:Collectively, these findings strongly suggest that selinexor is a promising therapeutic agent for TNBC as a single agent and in combination with standard chemotherapy.
Project description:PURPOSE:To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models. EXPERIMENTAL DESIGN:We established 12 PDXs from BRAF inhibitor-progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches. RESULTS:We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals. CONCLUSIONS:Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. See related commentary by Hartsough and Aplin, p. 1550.
Project description:Improved prognosis for triple-negative breast cancer (TNBC) has currently plateaued and the development of novel therapeutic strategies is required. Therefore, we aimed to explore the anti-tumor effect of eribulin and paclitaxel combination therapy for TNBC. The effect of eribulin and paclitaxel in combination was tested, with both concurrent and sequential administration, using four TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-157, and Mx-1) in vitro and in an MDA-MB-231 BALB/c-nu/nu mouse xenograft model. The expression of epithelial-mesenchymal phenotypic markers was analyzed by western blotting and immunohistochemical analyses. Simultaneous administration of eribulin and paclitaxel resulted in a synergistic anti-tumor effect with MDA-MB-231 and Hs578T cells, but not MDA-MB-157 and Mx-1 cells, in vitro. Moreover, pre-treatment with one drug significantly enhanced sensitivity to the subsequently administrated second drug in MDA-MB-231 and Hs578T cells. Eribulin increased E-cadherin expression and decreased the expression of mesenchymal markers in MDA-MB-231 and Hs578T cells. In contrast, paclitaxel increased the expression of mesenchymal markers. When epithelial-mesenchymal transition was induced by TGF-?1, eribulin sensitivity was enhanced. In contrast, a TGF-? receptor kinase inhibitor decreased eribulin sensitivity. In MDA-MB-231 tumor-bearing mice, concurrent administration of low doses of eribulin and paclitaxel significantly inhibited tumor growth compared to that with either monotherapy. Moreover, single administration of eribulin before the initiation of paclitaxel treatment decreased vimentin expression and reduced the average tumor volume in a mouse xenograft model. Eribulin and paclitaxel show synergistic anti-tumor effect by altering the epithelial-mesenchymal phenotype. This combination therapy could represent a novel therapeutic strategy for TNBC.