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H.A. Drabkin
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MINI 09 - Drug Resistance (ID 107)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:L. Villaruz, J. Minna
- Coordinates: 9/07/2015, 16:45 - 18:15, 205+207
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MINI09.13 - Neuropilin-2 Promotes Acquired Resistance to EGFR-TKI Associated with the Epithelial–Mesenchymal Transition in Lung Cancer (ID 1271)
17:55 - 18:00 | Author(s): H.A. Drabkin
- Abstract
Background:
Lung cancer accounts for one-fifth of cancer deaths worldwide with invasion, metastases and drug resistance representing major causes of mortality and barriers to cure. While lung cancers with activating mutations in the EGF receptor (EGFR) are susceptible to tyrosine kinase inhibitors (TKI), such as erlotinib and gefitinib, the efficacy of these agents is limited by the inevitable development of resistance. The epithelial-mesenchymal transition (EMT), by which epithelial cells acquire a mesenchymal and invasive phenotype, is one mechanism promoting EGFR-TKI resistance, including resistance to 3[rd] generation T790M-specific inhibitors. However, the molecular connections between EMT and resistance are not well understood. Here we report that upregulation of Neuropilin-2 (NRP2) is crucial for development of EGFR-TKI resistance associated with the EMT. NRP2 is a cell surface receptor for SEMA3F, a secreted semaphorin with tumor suppressor activity that is down-regulated during EMT. NRP1 and NRP2 are also co-receptors and signaling enhancers for several growth-promoting ligands such as VEGF, HGF and FGF. We previously reported that NRP2 was induced by TGFβ as part of an EMT response in lung cancers and that NRP2 knockdown suppressed the EMT phenotype, including local tumor invasion in a subcutaneous xenograft model.
Methods:
Immunohistochemistry (IHC) was performed for NRP2 on patient biopsies, before and after development of gefitinib resistance. EGFR mutant NSCLC cell lines, transfected with control or NRP2-specific shRNAs, were selected for gefitinib/erlotinib resistance in vitro, using progressively increasing concentrations or continuous exposure to IC~50~ levels of EGFR TKIs. Western blot analysis confirmed changes in NRP2 expression along with selected markers of EMT. MTS viability assays determined drug sensitivity while migration and invasion were assessed using Boyden chambers. Growth as spheroids was assessed in 1% methylcellulose medium in low-adherence plates.
Results:
Increased NRP2 was observed in lung tumor biopsies following acquisition of EMT-associated gefitinib-resistance, and in HCC4006-ER cells, which acquired a stable erlotinib-resistant EMT phenotype. In vitro, using multiple EGFR mutant cell lines, NRP2 knockdown blocked acquired gefitinib-resistance, arising both spontaneously following growth in IC~50~ concentrations or after exposure to TGFβ. Of interest, spontaneously-resistant cells exhibited increased migration similar to cells stimulated with TGFβ. NRP2 knockdown also blocked tumorsphere formation, which has been associated with stem-cell characteristics and drug resistance.
Conclusion:
Collectively, our results demonstrate that NRP2 is a mediator of acquired EGFR-TKI resistance. The results also suggest that NRP2 blocking antibodies could be useful for enhancing the duration of response to EGFR inhibitors, including those targeting the T790M mutation.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-055 - Anti-Glut-1 Antibody as a Novel Therapeutic Modality against Breast and Lung Cancers (ID 3158)
09:30 - 09:30 | Author(s): H.A. Drabkin
- Abstract
Background:
The growth and survival of many tumors are dependent upon high glucose uptake to meets it energy needs. A family of glucose transporters proteins (GLUTs) facilitate glucose uptake by cancer cells. There are at least 12 known isoforms of glucose transporter proteins. These transporter proteins differ in their kinetics and its expression is tailored to the requirement of the individual cell type. Although more than one Glut transporter protein may be expressed by a particular tumor cell type, tumors frequently over express Glut-1 which is a high affinity glucose transporter protein allowing the tumor to internalized a relatively large amount of glucose. Indeed tumoral Glut-1 expression correlates with the intensity of glucose uptake seen in a PET scan. We have previously demonstrated that anti-Glut-1 monoclonal antibody inhibited proliferation and induced apoptosis in breast cancer and lung cancer cell lines in vitro. Here we report the results of our in vivo studies where we investigated the ability of anti-Glut-1 monoclonal antibody to retard tumor growth in orthotopically implanted MDAMB-231 cell line in female athymic nude mice. We also examined the ability of the Glut-1 antibody to augment the retardation of tumor growth induced by cisplatin, paclitaxel, tamoxifen, and trastuzumab in the study.
Methods:
MDA-MB-231 breast cancer cells were orthotopically implanted in female thymic nude mice. Cohorts of tumor bearing mice were treated with control solution (PBS) or different dose levels of anti-Glut 1 antibody through tail vein injections. The Glut-1 monoclonal antibody used in the studies detailed here was generated from the clone SPM498. Once an optimal dose of Glu-1 antibody was selected we tested its ability to augment the growth retardation induced by cisplatin, paclitaxel, tamoxifen and trastuzumab. Tumors were measured as treatments continued. At the sign of earliest distress the animals were sacrificed and the organs were harvested and examined for evidence of toxicity and metastases. The harvested tumors were then subjected to Western blot and immunohistochemical analysis to look for markers of apoptosis and proliferation. All the organs and peripheral blood were examined to look for evidence of organ toxicity as a consequence of treatment by the Glut-1 antibody.
Results:
Anti-Glut 1 antibody can be administered safely in high doses to mice. No consistent organ toxicities associated with Glut-1 treatment were observed. Specifically, there was no central nervous system side effects noted in the mice given that the brain accounts for approximately 30% of the total glucose consumption. Treatment with Anti-Glut-1 antibody did not demonstrate significant single agent activity; however an increase in survival was observed in mice treated with the combination of tamoxifen and the anti-Glut-1 antibody compared with tamoxifen alone. The results of the detailed analyses will be presented at the meeting.
Conclusion:
Our studies demonstrate that anti-Glut1 antibody can be safely administered to mice without major organ toxicity including CNS toxicity. It demonstrated limited anti-tumor efficacy as a single agent, but it shows an increased anti-tumor effect when combined with tamoxifen. Further studies evaluating the combination of anti-Glut-1 antibody with targeted and hormonal agents are warranted.
