Abstract The epithelial-to-mesenchymal transition (EMT) is an embryonic process that becomes latent in most normal adult tissues. Recently, we have shown that induction of EMT endows breast epithelial cells with stem cell traits. In this report, we have further characterized the EMT-derived cells and shown that these cells are similar to mesenchymal stem cells (MSCs) with the capacity to differentiate into multiple tissue lineages. For this purpose, we induced EMT by ectopic expression of Twist, Snail, or transforming growth factor-β in immortalized human mammary epithelial cells. We found that the EMT-derived cells and MSCs share many properties including the antigenic profile typical of MSCs, that is, CD44+, CD24−, and CD45−. Conversely, MSCs express EMT-associated genes, such as Twist, Snail, and mesenchyme forkhead 1 (FOXC2). Interestingly, CD140b (platelet-derived growth factor receptor-β), a marker for naive MSCs, is exclusively expressed in EMT-derived cells and not in their epithelial counterparts. Moreover, functional analyses revealed that EMT-derived cells but not the control cells can differentiate into alizarin red S-positive mature osteoblasts, oil red O-positive adipocytes and alcian blue-positive chondrocytes similar to MSCs. We also observed that EMT-derived cells but not the control cells invade and migrate towards MDA-MB-231 breast cancer cells similar to MSCs. In vivo wound homing assays in nude mice revealed that the EMT-derived cells home to wound sites similar to MSCs. In conclusion, we have demonstrated that the EMT-derived cells are similar to MSCs in gene expression, multilineage differentiation, and ability to migrate towards tumor cells and wound sites.
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.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).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).Collectively, these findings strongly suggest that selinexor is a promising therapeutic agent for TNBC as a single agent and in combination with standard chemotherapy.
<div>AbstractPurpose:<p>Cyclin E (<i>CCNE1</i>) has been proposed as a biomarker of sensitivity to adavosertib, a Wee1 kinase inhibitor, and a mechanism of resistance to HER2-targeted therapy.</p>Experimental Design:<p>Copy number and genomic sequencing data from The Cancer Genome Atlas and MD Anderson Cancer Center databases were analyzed to assess <i>ERBB2</i> and <i>CCNE1</i> expression. Molecular characteristics of tumors and patient-derived xenografts (PDX) were assessed by next-generation sequencing, whole-exome sequencing, fluorescent <i>in situ</i> hybridization, and IHC. <i>In vitro, CCNE1</i> was overexpressed or knocked down in HER2+ cell lines to evaluate drug combination efficacy. <i>In vivo</i>, NSG mice bearing PDXs were subjected to combinatorial therapy with various treatment regimens, followed by tumor growth assessment. Pharmacodynamic markers in PDXs were characterized by IHC and reverse-phase protein array.</p>Results:<p>Among several <i>ERBB2</i>-amplified cancers, <i>CCNE1</i> co-amplification was identified (gastric 37%, endometroid 43%, and ovarian serous adenocarcinoma 41%). We hypothesized that adavosertib may enhance activity of HER2 antibody–drug conjugate trastuzumab deruxtecan (T-DXd). <i>In vitro</i>, sensitivity to T-DXd was decreased by cyclin E overexpression and increased by knockdown, and adavosertib was synergistic with topoisomerase I inhibitor DXd. <i>In vivo</i>, the T-DXd + adavosertib combination significantly increased γH2AX and antitumor activity in HER2 low, cyclin E amplified gastroesophageal cancer PDX models and prolonged event-free survival (EFS) in a HER2-overexpressing gastroesophageal cancer model. T-DXd + adavosertib treatment also increased EFS in other HER2-expressing tumor types, including a T-DXd–treated colon cancer model.</p>Conclusions:<p>We provide rationale for combining T-DXd with adavosertib in HER2-expressing cancers, especially with co-occuring <i>CCNE1</i> amplifications.</p></div>
Abstract Purpose: Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor that is FDA approved for HER2-overexpressing/amplified (HER2+) breast cancer. In this preclinical study, we explored the efficacy of neratinib in combination with inhibitors of downstream signaling in HER2+ cancers in vitro and in vivo. Experimental Design: Cell viability, colony formation assays, and Western blotting were used to determine the effect of neratinib in vitro. In vivo efficacy was assessed with patient-derived xenografts (PDX): two breast, two colorectal, and one esophageal cancer (with HER2 mutations). Four PDXs were derived from patients who received previous HER2-targeted therapy. Proteomics were assessed through reverse phase protein arrays and network-level adaptive responses were assessed through Target Score algorithm. Results: In HER2+ breast cancer cells, neratinib was synergistic with multiple agents, including mTOR inhibitors everolimus and sapanisertib, MEK inhibitor trametinib, CDK4/6 inhibitor palbociclib, and PI3Kα inhibitor alpelisib. We tested efficacy of neratinib with everolimus, trametinib, or palbociclib in five HER2+ PDXs. Neratinib combined with everolimus or trametinib led to a 100% increase in median event-free survival (EFS; tumor doubling time) in 25% (1/4) and 60% (3/5) of models, respectively, while neratinib with palbociclib increased EFS in all five models. Network analysis of adaptive responses demonstrated upregulation of EGFR and HER2 signaling in response to CDK4/6, mTOR, and MEK inhibition, possibly providing an explanation for the observed synergies with neratinib. Conclusions: Taken together, our results provide strong preclinical evidence for combining neratinib with CDK4/6, mTOR, and MEK inhibitors for the treatment of HER2+ cancer.
Abstract Background: Phosphatase and tensin homologue (PTEN) loss is a frequently disrupted tumor suppressor in cancer. PTEN serves as a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway to promote balanced cell proliferation, survival and differentiation. Consequently, loss of PTEN function increases cell proliferation, decreases cell death and contributes to tumor initiation. PTEN loss occurs across a variety of cancer subtypes, and these PTEN deficient tumors are dependent on PI3Kβ activity. As a result, therapeutic approaches that focus on the inhibition of PI3Kβ isoform in PTEN-null cells are of interest. We evaluate the efficacy of PI3Kβ inhibitors focused on triple negative breast cancers with loss of PTEN function. Methods: We studied the effects of p110β inhibitor AZD8186 in a panel of ten TNBC cell lines, which included four PTEN-mutant cell lines. Three of four cell lines were confirmed to have PTEN loss on western blot. In vitro, cell proliferation assays were performed to determine the half maximal inhibitory concentration (IC50) after 3 days of treatment and to test the effects in combination with chemotherapy. We confirmed the sensitivity of the PTEN-null cell lines to AZD8186 with colony formation assays. Western blot analysis was performed to assess PTEN expression and PI3K pathway activation in MDA-MB-436 and MDA-MB-468 cell lines, and to evaluate effects of AZD8186 on PI3K signaling. Results: Cell lines with PTEN loss were significantly more sensitive to AZD8186 in vitro (p= 0.008). AZD8186 inhibited PI3K signaling. Western blot revealed decreased activation of pAKT, pGSK3β, pPRAS40 and pS6. Activation of other pathways were evaluated with no activation of the MAPK/ERK or MEK pathways appreciated. AZD8186 treatment resulted in increased apoptosis but did not have a significant effect on cell-cycle progression in PTEN-deficient cell lines. AZD8186 was not synergistic with eribulin, paclitaxel or carboplatin; combinations with novel targeted therapies in vitro and in vivo are ongoing. Conclusion: AZD8186 had single agent efficacy in PTEN-deficient triple negative breast cancer cell lines in vitro. Further study is needed to identify rationale combinations and in vivo efficacy. Citation Format: Nicci Owusu-Brackett, Ming Zhao, Argun Akcakanat, Kurt W. Evans, Erkan Yuca, Funda Meric-Bernstam. Efficacy of PI3Kβ inhibitor AZD8186 in PTEN-deficient triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5802.
<p>Supplementary Figure 1: HER2 status of patients and corresponding PDX models. HER2 expression was assessed by IHC and ERBB2 amplification status by FISH and NGS. FISH, fluorescence in situ hybridization; HER2, human epidermal growth factor receptor 2; NGS, next-generation sequencing. Alterations represents genomic alterations in available CLIA genomic testing on archival patient samples (matching PDX hosts). CLIA results were not available for patients matching PDX.003.025, PDX.003.263, and PDX.003.230.</p><p>Supplementary Figure 2: Actionable gene alterations in patient biopsies compared to the alteration in the resulting PDX as determined by whole exome sequencing on both.</p><p>Supplementary Figure 3: Cell viability assay with crizotinib. Cells were treated with crizotinib at serial dilutions for 72 hours. Following SRB staining, IC50 was calculated using CalcuSyn.</p><p>Supplementary Figure 4. In vivo assessment of zanidatamab in combination with BET inhibitor JQ1. 50 mg/kg JQ1 had no antitumor effects as single agent and did not add benefit when combined with 16 mg/kg zanidatamab.</p>
<div>Abstract<p>Zanidatamab is a bispecific human epidermal growth factor receptor 2 (HER2)-targeted antibody that has demonstrated antitumor activity in a broad range of HER2-amplified/expressing solid tumors. We determined the antitumor activity of zanidatamab in patient-derived xenograft (PDX) models developed from pretreatment or postprogression biopsies on the first-in-human zanidatamab phase I study (NCT02892123). Of 36 tumors implanted, 19 PDX models were established (52.7% take rate) from 17 patients. Established PDXs represented a broad range of HER2-expressing cancers, and <i>in vivo</i> testing demonstrated an association between antitumor activity in PDXs and matched patients in 7 of 8 co-clinical models tested. We also identified amplification of <i>MET</i> as a potential mechanism of acquired resistance to zanidatamab and demonstrated that MET inhibitors have single-agent activity and can enhance zanidatamab activity <i>in vitro</i> and <i>in vivo</i>. These findings provide evidence that PDXs can be developed from pretreatment biopsies in clinical trials and may provide insight into mechanisms of resistance.</p>Significance:<p>We demonstrate that PDXs can be developed from pretreatment and postprogression biopsies in clinical trials and may represent a powerful preclinical tool. We identified amplification of <i>MET</i> as a potential mechanism of acquired resistance to the HER2 inhibitor zanidatamab and MET inhibitors alone and in combination as a therapeutic strategy.</p><p><i><a href="https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-14-5-ITI" target="_blank">This article is featured in Selected Articles from This Issue, p. 695</a></i></p></div>
