Marco Zanotto

An FGFR3 autocrine loop sustains acquired resistance to trastuzumab in gastric cancer patients

Purpose: The majority of gastric cancer patients who achieve an initial response to trastuzumab-based regimens develop resistance within 1 year of treatment. This study was aimed at identifying the molecular mechanisms responsible for resistance. Experimental Design: A HER2+-trastuzumab sensitive NCI-N87 gastric cancer orthotopic nude mouse model was treated with trastuzumab until resistance emerged. Differentially expressed transcripts between trastuzumab-resistant and sensitive gastric cancer cell lines were annotated for functional interrelatedness by Ingenuity Pathway Analysis software. Immunohistochemical analyses were performed in pretreatment versus posttreatment biopsies from gastric cancer patients receiving trastuzumab-based treatments. All statistical tests were two-sided. Results: Four NCI-N87 trastuzumab-resistant (N87-TR) cell lines were established. Microarray analysis showed HER2 downregulation, induction of epithelial-to-mesenchymal transition, and indicated fibroblast growth factor receptor 3 (FGFR3) as one of the top upregulated genes in N87-TR cell lines. In vitro, N87-TR cell lines demonstrated a higher sensitivity than did trastuzumab-sensitive parental cells to the FGFR3 inhibitor dovitinib, which reduced expression of pAKT, ZEB1, and cell migration. Oral dovitinib significantly (P = 0.0006) reduced tumor burden and prolonged mice survival duration in N87-TR mouse models. A higher expression of FGFR3, phosphorylated AKT, and ZEB1 were observed in biopsies from patients progressing under trastuzumab-based therapies if compared with matched pretreatment biopsies. Conclusions: This study identified the FGFR3/AKT axis as an escape pathway responsible for trastuzumab resistance in gastric cancer, thus indicating the inhibition of FGFR3 as a potential strategy to modulate this resistance. Clin Cancer Res; 22(24); 6164–75. ©2016 AACR.

Combined inhibition of IL1, CXCR1/2, and TGFβ signaling pathways modulates in-vivo resistance to anti-VEGF treatment.

Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin-1 (IL1), CXC receptors (CXCR)1/2 ligands, and transforming growth factor &bgr; (TGF&bgr;) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the antivascular endothelial growth factor (anti-VEGF) antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse this anti-VEGF resistance. Bevacizumab-resistant FGBR pancreatic cancer cells were treated in vitro with bevacizumab, the recombinant human IL1 receptor antagonist anakinra, the monoclonal antibody against TGF&bgr; receptor type II TR1, and a novel recombinant antibody binding CXCR1/2 ligands. The FGBR cells treated with these agents in combination had significantly higher levels of E-cadherin and lower levels of vimentin, IL6, phosphorylated p65, and SMAD2, and showed significantly lower migration rates than did their controls treated with the same agents without bevacizumab or with a single agent bevacizumab as a control. Consistently, the combination of these agents with bevacizumab reduced the FGBR tumor burden and significantly prolonged mice survival compared with bevacizumab in monotherapy. Tumors from mice receiving the combination treatment showed significantly lower expression of IL6 and phosphorylated SMAD2, higher expression of E-cadherin and lower levels of vimentin, and a significantly lower infiltration by CD11b+ cells compared with bevacizumab-treated controls. This study suggests that inhibition of IL1, CXCR1/2, and TGF&bgr; signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing epithelial–mesenchymal transition and inhibiting CD11b+ proangiogenic myeloid cells’ tumor infiltration.

TAK1-regulated expression of BIRC3 predicts resistance to preoperative chemoradiotherapy in oesophageal adenocarcinoma patients

Background:About 20% of resectable oesophageal carcinoma is resistant to preoperative chemoradiotherapy. Here we hypothesised that the expression of the antiapoptotic gene Baculoviral inhibitor of apoptosis repeat containing (BIRC)3 induced by the transforming growth factor β activated kinase 1 (TAK1) might be responsible for the resistance to the proapoptotic effect of chemoradiotherapy in oesophageal carcinoma.Methods:TAK1 kinase activity was inhibited in FLO-1 and KYAE-1 oesophageal adenocarcinoma cells using (5Z)-7-oxozeaenol. The BIRC3 mRNA expression was measured by qRT–PCR in 65 pretreatment frozen biopsies from patients receiving preoperatively docetaxel, cisplatin, 5-fluorouracil, and concurrent radiotherapy. Receiver operator characteristic (ROC) analyses were performed to determine the performance of BIRC3 expression levels in distinguishing patients with sensitive or resistant carcinoma.Results:In vitro, (5Z)-7-oxozeaenol significantly reduced BIRC3 expression in FLO-1 and KYAE-1 cells. Exposure to chemotherapeutic agents or radiotherapy plus (5Z)-7-oxozeaenol resulted in a strong synergistic antiapoptotic effect. In patients, median expression of BIRC3 was significantly (P<0.0001) higher in adenocarcinoma than in the more sensitive squamous cell carcinoma subtype. The BIRC3 expression significantly discriminated patients with sensitive or resistant adenocarcinoma (AUC-ROC=0.7773 and 0.8074 by size-based pathological response or Mandards tumour regression grade classifications, respectively).Conclusions:The BIRC3 expression might be a valid biomarker for predicting patients with oesophageal adenocarcinoma that could most likely benefit from preoperative chemoradiotherapy.

Homeobox B9 mediates resistance to anti-VEGF therapy in colorectal cancer patients

Purpose: The identification of predictive biomarkers for antiangiogenic therapies remains an unmeet need. We hypothesized that the transcription factor Homeobox B9 (HOXB9) could be responsible for the tumor resistance to the anti-VEGF agent bevacizumab. Experimental Design: HOXB9 expression and activation were measured in eight models of colorectal and pancreatic cancer with different resistance to bevacizumab. Serum levels of Angiopoietin-like Protein (Angptl)2, CXC receptor ligand (CXCL)1, IL8, and TGFβ1 in tumor-bearing mice were measured by multiplex xMAP technology. HOXB9 expression was measured by immunohistochemical analysis in 81 pretreatment specimens from metastatic colorectal cancer patients. Differences in progression-free survival (PFS) were determined using a log-rank test. Results: HOXB9-positive tumors were resistant to bevacizumab, whereas mice bearing HOXB9-negative tumors were cured by this agent. Silencing HOXB9 in bevacizumab-resistant models significantly (P < 0.05) reduced Angptl2, CXCL1, IL8, and TGFβ1 levels, reverted their mesenchymal phenotype, reduced CD11b+ cells infiltration, and restored, in turn, sensitivity to bevacizumab. HOXB9 had no prognostic value in patients treated with a first-line chemotherapeutic regimen noncontaining bevacizumab. However, patients affected by an HOXB9-negative tumor had a significantly longer PFS compared with those with an HOXB9-positive tumor if treated with a first-line regimen containing bevacizumab (18.0 months vs. 10.4 months; HR 2.037; 95% confidence interval, 1.006–4.125; P = 0.048). Conclusions: These findings integrate the complexity of numerous mechanisms of anti-VEGF resistance into the single transcription factor HOXB9. Silencing HOXB9 could be a promising approach to modulate this resistance. Our results candidate HOXB9 as predictive biomarker for selecting colorectal cancer patients for antiangiogenic therapy. Clin Cancer Res; 23(15); 4312–22. ©2017 AACR.

Toll-Like Receptor 9 Agonists for Cancer Therapy

The immune system has acquired increasing importance as a key player in cancer maintenance and growth. Thus, modulating anti-tumor immune mediators has become an attractive strategy for cancer treatment. Toll-like receptors (TLRs) have gradually emerged as potential targets of newer immunotherapies. TLR-9 is preferentially expressed on endosome membranes of B-cells and plasmacytoid dendritic cells (pDC) and is known for its ability to stimulate specific immune reactions through the activation of inflammation-like innate responses. Several synthetic CpG oligonucleotides (ODNs) have been developed as TLR-9 agonists with the aim of enhancing cancer immune surveillance. In many preclinical models, CpG ODNs were found to suppress tumor growth and proliferation both in monotherapy and in addition to chemotherapies or target therapies. TLR-9 agonists have been also tested in several clinical trials in patients with solid tumors. These agents showed good tolerability and usually met activity endpoints in early phase trials. However, they have not yet been demonstrated to significantly impact survival, neither as single agent treatments, nor in combination with chemotherapies or cancer vaccines. Further investigations in larger prospective studies are required.

Abstract 3265: Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors

Resistance to anti-angiogenic therapies poses one of the greatest challenges in gastrointestinal tumors research. We recently identified several proinflammatory secreted factors, including interleukin-1 (IL1), CXC ligand (CXCL) 1 and 8, Transforming Growth Factor β (TGFβ)1, and Angiopoietin-like Protein 2 (ANGPTL2), that were overexpressed in murine models of tumors resistant to the anti-VEGF antibody bevacizumab (BEV). These factors induced epithelial-to-mesenchymal transition (EMT) and increased aggressiveness. HOXB9 has been identified as a key transcription factor in common for these factors. Here, we hypothesized that HOXB9 might be responsible for BEV resistance in gastrointestinal tumors. HOXB9 expression and activation were measured in BEV-sensitive COLO357FG and in their BEV-resistant counterpart FGBR pancreatic cancer cell lines, and in LOVO, MDST8, LIM2099, CCK81, GP5D, and SNUC4 colon cancer cell lines by EMSA and DAPA. Serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 in nude mice bearing tumors were measured by multiplex xMAP technology. Immunohistochemical analyses were performed in pre- vs. post-progression biopsies from colorectal cancer patients receiving BEV. HOXB9 protein was more expressed and activated in FGBR than did in COLO357FG cells. shRNA to knock down HOXB9 significantly (P In conclusion, we identified HOXB9 as crucial transcription factor to sustain BEV resistance in gastrointestinal tumors. Silencing of HOXB9 is a promising approach to modulate this resistance. Our results candidate HOXB9 as potential biomarker for selecting patients with colorectal and pancreatic cancer for antiangiogenic therapy. Citation Format: Carmine Carbone, Geny Piro, Francesca Simionato, Fotios Loupakis, Chiara Cremolini, Gabriella Fontanini, Federica Di Nicolantonio, Elisa Moratti, Francesca Ligorio, Marco Zanotto, Raffaela Santoro, Maria Mihaela Mina, Aldo Scarpa, Alberto Bardelli, Giampaolo Tortora, Davide Melisi. Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3265.

Abstract 579: TAK1-regulated expression of BIRC3 is responsible for chemoradiotherapy (CRT) resistance in esophagogastric junction (EGJ) adenocarcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Preoperative CRT followed by surgery is the most common approach for patients with resectable esophageal and EGJ cancers. Based on the histology, patients with adenocarcinoma consistently demonstrated significantly lower rate of pathological complete response if compared with patients with squamous cell carcinoma, thus the need for accurate biomarkers to select the patients with esophageal and EGJ adenocarcinoma most likely to benefit from preoperative CRT has become even more critical. We recently demonstrated that the MAP3K TGF-βeta-activated kinase-1 (TAK1) is responsible for the resistance to the proapoptotic effect of chemotherapeutic agents by increasing the transcription of the member of the inhibitor of apoptosis proteins family BIRC3 in preclinical models of pancreatic cancer. Here, we hypothesized that the TAK1-regulated expression of BIRC3 might be responsible for the resistance to CRT in EGJ adenocarcinoma. Materials and methods: TAK1 kinase activity was targeted in FLO-1 and KYAE-1 esophageal cells by using (5Z)-7oxozeaenol. To test the effect of reducing BIRC3 expression on the resistance to CRT, FLO-1 and KYAE-1 cells were treated with increasing doses of cisplatin, 5-fluorouracil, paclitaxel, or radiotherapy in combination with (5Z)-7oxozeaenol. Drug interactions were studied for synergism according to Chou and Talalay method. Apoptotic induction was studied by western blot analysis of PARP and caspase 3 cleavages as well as by AnnexinV staining. BIRC3 expression was measured in 33 pretreatment biopsies from patients with EGJ adenocarcinoma and 34 from patients with esophageal squamous cell carcinoma receiving neoadjuvant CRT by Real-Time PCR. Tumor response was evaluated by Tumour regression grade (TRG) and by Size-based Pathological Response (SPR) scores. Correlation between BIRC3 expression and treatment response was analysed by ROC curve analysis. Results: In vitro, (5Z)-7oxozeaenol significantly reduced BIRC3 expression in FLO-1 and KYAE-1 esophageal cells. Exposure to sublethal equitoxic doses of chemotherapeutic agents plus (5Z)-7oxozeaenol pretreatment resulted in a strong synergistic anti-proliferative effect. Baseline expression of BIRC3 was significantly higher in patients with EGJ adenocarcinoma if compared with the more sensitive squamous-cell carcinoma subtype. Moreover, patients with EGJ adenocarcinoma expressing higher pretreatment levels of BIRC3 had a significantly poorer treatment response than did those with lower expression, indicating that BIRC3 expression significantly correlates with response to preoperative CRT (AUC-ROC = 0.777 and 0.807 for SPR and TRG, respectively). Conclusions: TAK1-regulated expression of BIRC3 might be a valid biomarker to predict resistance to CRT in EGJ adenocarcinoma patients. Citation Format: Geny Piro, Simone Giacopuzzi, Maria Bencivenga, Carmine Carbone, Giuseppe Verlato, Melissa Frizziero, Maria Mihaela Mina, Marco Zanotto, Valeria Merz, Giovanni De Manzoni, Giampaolo Tortora, Davide Melisi. TAK1-regulated expression of BIRC3 is responsible for chemoradiotherapy (CRT) resistance in esophagogastric junction (EGJ) adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 579. doi:10.1158/1538-7445.AM2015-579

Abstract 3605: Combined inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways modulates in vivo acquired resistance to anti-VEGF treatment

Introduction: Resistance of tumors to antiangiogenic therapies is becoming increasingly relevant. We recently identified interleukin (IL)-1, CXC receptors (CXCR)1/2 ligands, and Transforming Growth Factor β (TGFβ) among the proinflammatory factors that were expressed at higher levels in murine models resistant to the anti-VEGF antibody bevacizumab. Here, we hypothesized that the combined inhibition of these proinflammatory signaling pathways might reverse the resistance to anti-VEGF treatment. Methods: Bevacizumab-resistant FGBR orthotopic tumor bearing mice received bevacizumab alone or in combination with a recombinant human IL-1 receptor antagonist, a monoclonal antibody against TGFβ Receptor type II, and a recombinant antibody binding CXCR1/2 ligands. Western blot analysis was performed to assess the activity of p65 NFB, Smad2 and on the expression of IL-6 in FGBR cells in culture. Immunohistochemical analysis of FFPE sections from FGBR tumors was performed to assess Smad2 phosphorylation and IL-6 expression. Wound healing assay was performed to assess cell motility in FGBR cells in culture. Immunohistochemical analysis was performed to assess the expression of E-Cadherin and Vimentin in FGBR tumors. Immunohistochemical analysis was performed to estimate the presence of Cd11b+ cells. Results: The combination of these agents with bevacizumab reduced the tumor burden and significantly prolonged mice survival if compared with single agent bevacizumab. Tumors from mice receiving the combination treatment demonstrated significantly lower expression of IL-6 and phosphorylation of p65 and Smad2 when compared with control. FGBR cells treated in vitro with the three agents plus bevacizumab had significantly higher levels of E-cadherin and lower levels of Vimentin, and exhibited significantly lower migration rates than did their bevacizumab- treated controls. Consistently, tumors from mice receiving the combination treatment demonstrated significantly higher expression of E-cadherin, lower levels of Vimentin, and a significantly lower infiltration by CD11b+ cells when compared with bevacizumab -treated controls. Conclusion: This study suggests that inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways is a potential therapeutic approach to modulate the acquired resistance to anti-VEGF treatment by reversing EMT and inhibiting CD11b+ proangiogenic myeloid cells tumor infiltration. Citation Format: Carmine Carbone, Anna Tamburrino, Geny Piro, Marco Zanotto, Maria Mihaela Mina, Silvia Zanini, Federico Boschi, Aldo Scarpa, Giampaolo Tortora, Davide Melisi. Combined inhibition of IL-1, CXCR1/2, and TGFβ signaling pathways modulates in vivo acquired resistance to anti-VEGF treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3605. doi:10.1158/1538-7445.AM2015-3605