Teresa C. Dugger

Blockade of TGF-β inhibits mammary tumor cell viability, migration, and metastases

TGF-βs are potent inhibitors of epithelial cell proliferation. However, in established carcinomas, autocrine/paracrine TGF-β interactions can enhance tumor cell viability and progression. Thus, we studied the effect of a soluble Fc:TGF-β type II receptor fusion protein (Fc:TβRII) on transgenic and transplantable models of breast cancer metastases. Systemic administration of Fc:TβRII did not alter primary mammary tumor latency in MMTV-Polyomavirus middle T antigen transgenic mice. However, Fc:TβRII increased apoptosis in primary tumors, while reducing tumor cell motility, intravasation, and lung metastases. These effects correlated with inhibition of Akt activity and FKHRL1 phosphorylation. Fc:TβRII also inhibited metastases from transplanted 4T1 and EMT-6 mammary tumors in syngeneic BALB/c mice. Tumor microvessel density in a mouse dorsal skin window chamber was unaffected by Fc:TβRII. Therefore, blockade of TGF-β signaling may reduce tumor cell viability and migratory potential and represents a testable therapeutic approach against metastatic carcinomas.

Human Breast Cancer Cells Selected for Resistance to Trastuzumab In vivo Overexpress Epidermal Growth Factor Receptor and ErbB Ligands and Remain Dependent on the ErbB Receptor Network

Purpose: We have investigated mechanisms of acquired resistance to the HER2 antibody trastuzumab in BT-474 human breast cancer cells. Experimental Design: BT-474 xenografts established in athymic nude mice were eliminated by trastuzumab. Continuous cell lines (HR for Herceptin resistant) were generated from tumors that recurred in the presence of continuous antibody therapy. Results: The isolated cells behaved resistant to trastuzumab in culture as well as when reinjected into nude mice. They retained HER2 gene amplification and trastuzumab binding and were exquisitely sensitive to peripheral blood mononuclear cells ex vivo in the presence of the antibody. The HR cells exhibited higher levels of phosphorylated epidermal growth factor receptor (EGFR) and EGFR/HER2 heterodimers. Phosphorylation of HER2 in HR cells was inhibited by the EGFR tyrosine kinase inhibitors erlotinib and gefitinib. Gefitinib also inhibited the basal association of p85 with phosphorylated HER3 in HR cells. Both inhibitors as well as the dual EGFR/HER2 inhibitor, lapatinib, induced apoptosis of the HR cells in culture. Growth of established HR5 xenografts was inhibited by erlotinib in vivo. In addition, the HR cells overexpressed EGFR, transforming growth factor α, heparin-binding EGF, and heregulin RNAs compared with the parental trastuzumab-sensitive cells. Conclusions: These results are consistent with the inability of trastuzumab to block the heterodimerization of HER2 and suggest that amplification of ligand-induced activation of ErbB receptors is a plausible mechanism of acquired resistance to trastuzumab that should be investigated in primary mammary cancers.

Inhibition of TGF-β with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression

We investigated whether TGF-beta induced by anticancer therapies accelerates tumor progression. Using the MMTV/PyVmT transgenic model of metastatic breast cancer, we show that administration of ionizing radiation or doxorubicin caused increased circulating levels of TGF-beta1 as well as increased circulating tumor cells and lung metastases. These effects were abrogated by administration of a neutralizing pan-TGF-beta antibody. Circulating polyomavirus middle T antigen-expressing tumor cells did not grow ex vivo in the presence of the TGF-beta antibody, suggesting autocrine TGF-beta is a survival signal in these cells. Radiation failed to enhance lung metastases in mice bearing tumors that lack the type II TGF-beta receptor, suggesting that the increase in metastases was due, at least in part, to a direct effect of TGF-beta on the cancer cells. These data implicate TGF-beta induced by anticancer therapy as a pro-metastatic signal in tumor cells and provide a rationale for the simultaneous use of these therapies in combination with TGF-beta inhibitors.

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Early changes in protein expression detected by mass spectrometry predict tumor response to molecular therapeutics.

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin β4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time- and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.

Inhibition of Mammalian Target of Rapamycin Is Required for Optimal Antitumor Effect of HER2 Inhibitors against HER2-Overexpressing Cancer Cells

Purpose: A significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mammalian target of rapamycin (mTOR) is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab. Experimental Design: Immunocompetent mice bearing HER2+ mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG positron emission tomography. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated. Results: Treatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26 of 26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phosphorylated S6 and Ki-67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phosphorylated S6 and growth in TSC2-expressing cells but not in TSC2-knockdown cells. Conclusions: Inhibition of PI3K and mTOR are required for the growth-inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer. (Clin Cancer Res 2009;15(23):7266–76)

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells.

We have studied the role of autocrine transforming growth factor-β (TGF-β) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-β activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-β receptors in both cell lines. Anti-pan-TGF-β antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-β receptors at the cell surface and exquisite sensitivity to exogenous TGF-β1, were transfected with a tetracycline-controllable dominant-negative TGF-βRII (ΔRII) construct. Although the TGF-β1 response was blocked by removal of tetracycline in MCF-7/ΔRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-β1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the ΔRII transgene product. In MCF-7 cells with a functional TGF-β signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-β1 unresponsive MCF-7 cells with high levels of TGF-βRII restored TGF-β1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-β signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.

Kinome-wide RNA interference screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition in ER-positive breast cancer

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR.

An ERBB1-3 Neutralizing Antibody Mixture With High Activity Against Drug-Resistant HER2+ Breast Cancers With ERBB Ligand Overexpression

Background Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance. Methods HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided. Results Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm 3 , SD = 924 mm 3 , vs Pan-HER mean = 565 mm 3 , SD = 499 mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts. Conclusions These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers.

Abstract P3-03-05: PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs)

Background: CDK4/6 inhibitors in combination with antiestrogens are approved for the treatment of ER+ advanced breast cancer. However, not all patients benefit from CDK4/6 inhibitors, underscoring the need to develop therapeutic strategies to circumvent de novo and acquired drug resistance. Methods: ER+ breast cancer cells (MCF-7, T47D, HCC1428, and HCC1500) were made resistant to increasing doses to the CDK4/6 inhibitor ribociclib (LEE011; Novartis). LEE011-resistant cells were characterized by 2D/3D growth, cell cycle, and immunoblot analyses. GSK2334470 (PDK1 inhibitor) and dinaciclib (CDK2 inhibitor) were used to modify resistance to ribociclib. PDK1 and pS6 immunohistochemistry (IHC) were performed on primary human tumor explants treated ex vivo with palbociclib. Results: Resistant cell lines (MCF-7/LR, T47D/LR, HCC1428/LR, and HCC1500/LR) exhibited an IC 50 at least 20-fold higher than that of their parental cells. They displayed cross-resistance to the CDK4/6 inhibitors palbociclib and abemaciclib. Immunoblot analysis of ribociclib-resistant cells showed increased levels of 3-phosphoinositide dependent protein kinase 1 (PDK1), S227 pRSK2 (target of PDK1), T308 pAKT (target of PDK1), and pS6 (downstream effector of the PDK1 target p70S6K), compared to parental drug sensitive cells. PDK1 is a master kinase that functions downstream of phosphoinositide 3-kinase (PI3K) and is crucial for the activation of AKT and many other AGC kinases including PKC, S6K, SGK, and RSK. Primary tumor explants treated ex vivo with palbociclib for 96 h also exhibited upregulation of PDK1 and pS6 by IHC. Cell cycle analysis revealed that CDK4/6 inhibition failed to induce G1 arrest, a reduction in S phase, and senescence in MCF-7/LR and T47D/LR compared to parental cells. Progression into S phase in the presence of ribociclib suggested upregulation of S-phase cyclins/CDKs. Indeed, the resistant cells exhibited significantly higher levels of pCDK2, cyclin A, cyclin E and S477/T479 pAKT, a CDK2-dependent phosphorylation of AKT required for full kinase activity and limited to the S-phase of the cell cycle. Pharmacological inhibition of PDK1 (with GSK2334470) or CDK2 (with dinaciclib) re-sensitized the ribociclib-resistant cells to CDK4/6 inhibitors. However, ribociclib/GSK2334470 inhibited MCF-7/LR and T47D/LR cell proliferation better than ribociclib/dinaciclib. Further, ribociclib/GSK2334470 but not ribociclib/dinaciclib completely abrogated pRb, pS6, pRSK2, pCDK2, cyclin A, and cyclin E, suggesting the PI3K/PDK1 pathway mediates acquired resistance to CDK4/6 inhibitors through dysregulation of the cell cycle. Consistent with these data, ribociclib/GSK2334470 inhibited growth of established MCF-7 xenografts in nude mice, significantly more potently than each drug alone. Conclusions: These data support a critical role for PI3K/PDK1 in acquired resistance to CDK4/6 inhibitors in ER+ breast cancer cells. Co-targeting of PI3K/PDK1 and CDK4/6 may overcome resistance to CDK4/6 inhibitors and is worthy of further translational and clinical investigation in patients with ER+ breast cancer. Citation Format: Jansen VM, Formisano L, Witkiewicz A, Estrada MV, Sanchez V, Dugger TC, Knudsen ES, Arteaga CL. PI3K/PDK1 mediates resistance to CDK4/6 inhibitors through dysregulation of S-phase cyclins/cyclin dependent kinases (CDKs) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-05.