Emin Ibrahimov

Prognostic gene-expression signature of carcinoma-associated fibroblasts in non-small cell lung cancer

The tumor microenvironment strongly influences cancer development, progression, and metastasis. The role of carcinoma-associated fibroblasts (CAFs) in these processes and their clinical impact has not been studied systematically in non-small cell lung carcinoma (NSCLC). We established primary cultures of CAFs and matched normal fibroblasts (NFs) from 15 resected NSCLC. We demonstrate that CAFs have greater ability than NFs to enhance the tumorigenicity of lung cancer cell lines. Microarray gene-expression analysis of the 15 matched CAF and NF cell lines identified 46 differentially expressed genes, encoding for proteins that are significantly enriched for extracellular proteins regulated by the TGF-β signaling pathway. We have identified a subset of 11 genes (13 probe sets) that formed a prognostic gene-expression signature, which was validated in multiple independent NSCLC microarray datasets. Functional annotation using protein–protein interaction analyses of these and published cancer stroma-associated gene-expression changes revealed prominent involvement of the focal adhesion and MAPK signaling pathways. Fourteen (30%) of the 46 genes also were differentially expressed in laser-capture–microdissected corresponding primary tumor stroma compared with the matched normal lung. Six of these 14 genes could be induced by TGF-β1 in NF. The results establish the prognostic impact of CAF-associated gene-expression changes in NSCLC patients.

Integrin α11β1 regulates cancer stromal stiffness and promotes tumorigenicity and metastasis in non-small cell lung cancer

Integrin α11β1 is a stromal cell-specific receptor for fibrillar collagens and is overexpressed in carcinoma-associated fibroblasts (CAFs). We have investigated its direct role in cancer progression by generating severe combined immune deficient (SCID) mice deficient in integrin α11 (α11) expression. The growth of A549 lung adenocarcinoma cells and two patient-derived non-small cell lung carcinoma (NSCLC) xenografts in these α11 knockout (α11−/−) mice was significantly impeded, as compared with wild-type (α11+/+) SCID mice. Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11−/− and α11+/+ mice showed significant reduction in the metastatic potential of these cells in the α11−/− mice. We identified that collagen cross-linking is associated with stromal α11 expression, and the loss of tumor stromal α11 expression was correlated with decreased collagen reorganization and stiffness. This study shows the role of integrin α11β1, a receptor for fibrillar collagen in differentiation of fibroblasts into CAFs. Furthermore, our data support an important role for α11 signaling pathway in CAFs, promoting tumor growth and metastatic potential of NSCLC cells and being closely associated with collagen cross-linking and the organization and stiffness of fibrillar collagen matrices.

Loss of canonical Smad4 signaling promotes KRAS driven malignant transformation of human pancreatic duct epithelial cells and metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.

Coiled-coil domain containing 68 (CCDC68) demonstrates a tumor-suppressive role in pancreatic ductal adenocarcinoma

Using integrative genomics and functional screening, we identified coiled-coil domain containing 68 (CCDC68) as a novel putative tumor suppressor gene (TSG) in pancreatic ductal adenocarcinoma (PDAC). CCDC68 allelic losses were documented in 48% of primary PDAC patient tumors, 50% of PDAC cell lines and 30% of primary patient derived xenografts. We also discovered a single nucleotide polymorphism (SNP) variant (SNP rs1344011) that leads to exon skipping and generation of an unstable protein isoform CCDC68Δ69–114 in 31% of PDAC patients. Overexpression of full length CCDC68 (CCDC68wt) in PANC-1 and Hs.766T PDAC cell lines lacking CDCC68 expression decreased proliferation and tumorigenicity in scid mice. In contrast, the downregulation of endogenous CCDC68 in MIAPaca-2 cells increased tumor growth rate. These effects were not observed with the deletion-containing isoform, CCDC68Δ69–114.

Abstract B282: The PLK-4 inhibitor CFI-400945 reduces tumor growth in patient-derived pancreatic xenografts.

The polo-like kinase 4 (PLK-4) has recently emerged as a target that strongly correlates with aggressive tumor growth, high levels of hypoxia and metastasis in patient-derived pancreatic xenografts. In contrast to other members of the PLK family, the role of PLK-4 in cancer remains mostly elusive. Overexpression of PLK-4 in tumors has been suggested to contribute to chromosomal instability due to its crucial role in centriole duplication and therefore presents an interesting molecular target for the development of new therapeutic options for pancreatic cancer patients. The newly developed PLK-4 inhibitor CFI-400945 has been shown to specifically target PLK-4, resulting in incorrect centriole duplication and is currently developed for clinical trials. We used 6 orthotopically implanted patient-derived pancreatic xenograft models with varying growth rates and levels of tumor hypoxia in order to investigate the efficiency of the PLK-4 inhibitor CFI-400945 as a mono-therapy or in combination with Gemcitabine. Animals were treated with CFI-400945 using a daily dose of 7.5mg/kg for 21 days for experiments evaluating activity as a mono-therapy or with 13.5mg/kg according to a 2-day-on-5-days-off schedule in combination bi-weekly 100mg/kg Gemcitabine for 21 days. Tumor weight after completion of the treatment and overall survival were evaluated. Immunofluorescence (IF) staining of xenograft sections was used to evaluate PLK-4 inhibition, tumor hypoxia and proliferation. Treatment with CFI-400945 as a mono-therapy for 21 days significantly reduced tumor weight in 4 of the 6 tested patient-derived pancreatic xenograft models and an increase in overall survival in a number of responsive models. The strongest response to CFI-400945 treatment was observed in the highly hypoxic fast growing xenograft models OCIP51 where medium survival was 103days in the treatment group when compared to 54 days in vehicle treated animals. Experiments investigating the benefit of the combination of CFI-400945 with Gemcitabine using a clinically relevant treatment schedule are currently in progress in 2 xenograft models and will be completed shortly. We furthermore examined the correlation between tumor hypoxia, proliferation and response to CFI-400945 treatment in order to establish biological correlates for treatment response. Preliminary results show that although tumor weight was reduced in most of the tested models, the strongest response to CFI-400945 as a mono-therapy was observed in highly hypoxic models when compared to medium or low hypoxic models. These findings suggest that the PLK-4 inhibitor CFI-400945 may provide an effective treatment strategy for pancreatic cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B282. Citation Format: Ines Lohse, Pinjiang Cao, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, David W. Hedley. The PLK-4 inhibitor CFI-400945 reduces tumor growth in patient-derived pancreatic xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B282.

Whole Genomes Define Concordance of Matched Primary, Xenograft, and Organoid Models of Pancreas Cancer

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among solid malignancies and improved therapeutic strategies are needed to improve outcomes. Patient-derived xenografts (PDX) and patient-derived organoids (PDO) serve as promising tools to identify new drugs with therapeutic potential in PDAC. For these preclinical disease models to be effective, they should both recapitulate the molecular heterogeneity of PDAC and validate patient-specific therapeutic sensitivities. To date however, deep characterization of PDAC PDX and PDO models and comparison with matched human tumour remains largely unaddressed at the whole genome level. We conducted a comprehensive assessment of the genetic landscape of 16 whole-genome pairs of tumours and matched PDX, from primary PDAC and liver metastasis, including a unique cohort of 5 ‘trios’ of matched primary tumour, PDX, and PDO. We developed a new pipeline to score concordance between PDAC models and their paired human tumours for genomic events, including mutations, structural variations, and copy number variations. Comparison of genomic events in the tumours and matched disease models displayed single-gene concordance across major PDAC driver genes, and genome-wide similarities of copy number changes. Genome-wide and chromosome-centric analysis of structural variation (SV) events revealed high variability across tumours and disease models, but also highlighted previously unrecognized concordance across chromosomes that demonstrate clustered SV events. Our approach and results demonstrate that PDX and PDO recapitulate PDAC tumourigenesis with respect to simple somatic mutations and copy number changes, and capture major SV events that are found in both resected and metastatic tumours.

Abstract B31: Establishment and molecular characterization of patient-derived tumor xenografts from resected tumors or ascites fluids of patients with pancreatic/ampullary/bile duct carcinomas

Pancreatic adenocarcinoma (PDAC) is the 4th most common cause of cancer deaths in North America, for both men and women with a 5-year survival rate of less than 5%. The poor prognosis rate is attributed to late presentation of the disease and the lack of effective treatment options. Large-scale genome sequencing efforts on PDAC tumors show evidence of high mutational burden and revealed a number of mutated genes affecting multiple oncogenic pathways. While there are significant endeavors in developing specific targeted agents against “driver” mutations, tumor diversity within and across patient population remains a key factor affecting therapeutic efficacy. In this context, the availability of large cohorts of genomically characterized patient-derived xenograft (PDX) tumor models may help to accelerate the development of novel therapies against this lethal cancer. PDX models provide a renewable resource to maintain a patient9s tumor ex vivo for pre-clinical or co-clinical studies. As part of The International Cancer Genome Consortium (ICGC), our laboratory has established 93 PDX models in non-obese diabetic and severe combined immune-deficient (NOD-SCID) mice from Whipple resection specimens. These tumors represent a heterogeneous group of neoplasms arising from the head, body and tail of pancreas, bile duct and Ampulla of Vater. All implantations including in the subcutaneous pocket at the flank or at the orthotopic pancreas site, were performed using 4-8 weeks old NOD-SCID mice. Successful growth and serial transplant to multiple mouse generations were observed in in 74 PDX models of the 93 implanted PDAC specimens, achieving an 80% engraftment rate, one of the highest reported in any type of cancer. Histology fidelity was preserved in the PDX models compared to corresponding patient tumors. Failed implants were due to specimens characterized by borderline malignancy and absence of tumor cells. Whole exome sequencing and copy number aberration profiling was completed for 61 PDXs and blood from the matched patients. Cancer-specific single nucleotide variation (SNV) load varied widely from 38 to 305 in PDXs. The most recurrent activating mutation was observed in KRAS with 77% of PDX models showing alterations at codon G12 (65%), G13 (8%) and Q61 (4%); in addition, 26% PDXs had a copy number gain in KRAS. Molecular comparisons of the 21 PDX models and their matched patient tumors showed that alternate allele frequency of KRAS mutation from exome sequencing of primary tumor is a strong indicator of the tumor cellularity; a higher tumor cellularity results in a larger overlap of cancer specific alterations between xenografts and corresponding patient tumors. We have demonstrated a successful establishment of PDX models that represent genomic architecture of major subclonal populations of patient PDAC primary tumors. Citation Format: Nikolina Radulovich, Emin Ibrahimov, Carson Holt, Vibha Raghavan, Tracy Zhao, Rob Denroch, Nhu-An Pham, Steve Gallinger, Melania Pintilie, Lincoln Stein, John McPherson, Lakshmi Muthuswamy, Ming Sound Tsao. Establishment and molecular characterization of patient-derived tumor xenografts from resected tumors or ascites fluids of patients with pancreatic/ampullary/bile duct carcinomas. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B31.

Abstract A102: Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts

Rationale: Using orthotopically-grown primary pancreatic cancer xenografts we recently identified an association between hypoxia and spontaneous metastasis formation, and we hypothesized that metastatic potential is conferred by the presence of tumor-initiating cells (TIC). We therefore investigated the relationship between hypoxia, tumor growth and TIC, and examined the effect of the hypoxia-activated prodrug TH-302 and ionizing radiation (IR) on pancreatic TIC and tumor growth. Methods: Eleven patient-derived pancreatic cancer xenograft models were used to examine tumor-initiation potential in relation to the tumor phenotype. To examine the effect of TH-302 and IR on TIC, tumor-bearing mice were treated with 50 mg/kg or 150 mg/kg TH-302, 10Gy of IR, or with the combination of TH-302 and IR. To examine the effect of TH-302 and IR on tumor growth, mice were treated with either TH-302 or IR alone, or with the combination treatment according to a clinically relevant schedule. Animals were treated with 50mg/kg TH-302 injected ip on days 1, 5 and 9 and/or 2Gy irradiation per day on days 2, 3 and 4. Tumor hypoxia was measured by EF5 administration and immunostaining. Results: TIC frequency varied across the patient-derived xenograft models and rapid tumor growth was strongly correlated with high TIC frequency but not hypoxia. Treatment with TH-302 in combination with IR led to a significant reduction in TIC frequency compared with either treatment alone in all models tested. Treatment-induced reduction in TIC frequency was independent of the magnitude of tumor hypoxia. Treatment-induced reduction of tumor growth was only observed in fast-growing hypoxic models. Additionally, TH-302 treatment also induced DNA damage in tumor tissue adjacent to the hypoxic zone which is consistent with the previously demonstrated bystander effect of TH-302. Conclusions: These results suggest that TIC can reside in both the oxic and the hypoxic microenvironment and that the combination of TH-302 and IR may present a novel therapeutic strategy to reduce the frequency of TIC and tumor growth rate of pancreatic tumors. Citation Format: Ines Lohse, Joanna Rasowski, Pinjian Cao, Melania Pintilie, Trevor Do, Emin Ibrahimov, Ming-Sound Tsao, Richard P. Hill, David W. Hedley. Combination treatment with hypoxia-activated prodrug TH-302 and radiation reduce pancreatic tumor initiating cells and tumor growth in patient-derived xenografts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A102.

Abstract A106: BRCA mutation sensitizes pancreatic tumors to treatment with cisplatin

Rationale: Recent clinical observations in pancreatic cancer suggest that cancers arising in germline BRCA1/2 mutation carriers are highly sensitive to Cisplatin chemotherapy. Both BRCA1 and BRCA2 are required for DNA double-strand break repair by homologous recombination (HR). Platinum chemotherapy generates interstrand cross-links that can only be adequately repaired by HR-based DNA repair, and consequently BRCA1/2-deficient cells are highly sensitive to platinum chemotherapy both in vitro and in vivo. With a strong biological rationale and favourable clinical observations, we aim to investigate whether BRCA mutation indeed sensitize to Cisplatin treatment and if the addition of the PARP inhibitor ABT-888 further reduces tumor growth in patient-derived pancreatic xenografts Methods: Primary xenografts were derived from three patients with known BRCA2 germline mutations (2 from surgical resections, 1 from ascites fluid). All 3 patients experienced a major response when treated with cisplatin. These models were used to assess the in vivo sensitivity to the drugs Cisplatin, Gemcitabine and the PARP inhibitor ABT-888, all of which are being used in the clinic. Treatment sensitivities were compared to 2 primary xenografts that were BRCA w/t. For single treatments, animals were treated with 4mg/kg Cisplatin weekly, 100mg/kg Gemcitabine biweekly or 12.5mg/kg ABT-888 daily for 30 days. For the combination treatment, animals were treated with a single dose of 4mg/kg Cisplatin followed by 30 days of 12.5mg/kg ABT-888 daily. Results: Preliminary results show no difference in tumor growth between BRCA2 wt and mt models was observed in response to treatment with ABT-888 alone. Treatment with cisplatin or gemcitabine on the other hand significantly reduced tumor growth and increased survival in all 3 BRCA2 mutated xenograft models, while being mostly ineffective in BRCA w/t tumors. The combination of Cisplatin and ABT-888 significantly reduced tumor growth when compared to ABT-888 alone. Conclusions: The results suggest that BRCA1/2 mt pancreatic xenograft models are highly sensitive to cisplatin. The availability of these models facilitates “near-clinical” testing of alternative strategies for optimizing the individualized treatment of patients carrying these rare mutations, and for the exploration of protocols incorporating novel agents. Citation Format: Ines Lohse, Pinjian Cao, Emin Ibrahimov, Ming-Sound Tsao, David W. Hedley. BRCA mutation sensitizes pancreatic tumors to treatment with cisplatin. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A106.

Abstract 2462: Coiled-coil domain 68 (CCDC68) plays a tumor suppressive role in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is driven by somatic alterations. Recent studies revealed that most genetic alterations are represented by rare mutations in many genes that are either drivers or passengers of PDAC. Using integrative genomics and functional screenings we identified coiled-coil domain 68 (CCDC68) as a putative tumor suppressor (TSG) in PDAC. Combination of Q-RT-PCR and FISH analysis revealed CCDC68 allelic losses in 50% of PDAC cell lines and 44% of primary patient xenografts. 21% of PDAC cell lines and primary patient xenografts also showed decreased levels of CCDC68 mRNA and protein. We also report the discovery of an unstable novel alternate splice variant (SNP rs. 1344011) of CCDC68 expressed by 30% of PDAC patients. Overexpression of full length CCDC68 (CCDC68wt) in PANC-1 and Hs.766T cell lines significantly decreased their cell proliferation, anchorage-independent growth and in vivo tumorigenicity in scid mice. However, the overexpression of novel transcript CCDC68 variant did not affect the cell proliferation or tumorigenicity potential of PANC-1 cells. Finally, we identified that CCDC68 negatively regulates MAPK signalling independently of KRAS status. In conclusion, CCDC68 loss-of-function through copy loss, mRNA downregulation and truncated protein expression suppresses the tumorigenicity of PDAC cell lines suggesting that CCDC68 is a candidate novel TSG in PDAC. Citation Format: Nikolina Radulovich, Lisa Leung, Emin Ibrahimov, Roya Navab, Shingo Sakashita, William Lockwood, Kelsie Thu, Yaroslav Fedeshyn, Jason Moffat, Wan Lam, Ming Tsao. Coiled-coil domain 68 (CCDC68) plays a tumor suppressive role in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2462. doi:10.1158/1538-7445.AM2014-2462