Tumors Of Colorectal Cancer Patients Research Articles

Association of oncogenic bacteria with colorectal cancer in South China.

To quantify Fusobacterium spp., Enterococcus faecalis (E.faecalis), Enterotoxigenic Bacteroides fragilis (ETBF), and Enteropathogenic Escherichia coli in colorectal cancer (CRC) patients and their possible association with CRC clinicopathogical features, we collected the resected tumors and adjacent normal tissues (N) from 97 CRC patients. 48 age- and sex-matched healthy controls (HC) were also recruited. Real-time PCR was used for bacterial quantification. The median abundance ofFusobacterium spp.(p < 0.001, vs. N; p < 0.01,vs. HC), E.faecalis (p < 0.05, vs. N; p < 0.01, vs. HC) and ETBF (p < 0.001, vs. N; p < 0.05,vs. HC) in tumor tissues was significantly higher than that detected in normal tissue and HC. E.faecalis was detected in 95.88% of tumors and 93.81% of adjacent tissues. Fusobacterium spp. was detected in 72.16% of tumors and 67.01% of adjacent tissues. The combined E.faecalis and Fusobacterium spp. were detected in 70.10% of tumors and 36.08% of adjacent normal tissues. All four bacteria were detected in 33.72% and 22.09% of paired tumor and adjacent normal tissues, respectively. E.faecalis and Fusobacterium spp. are enriched in both tumor and adjacent tissue of CRC patients when compared to HC, suggesting that it is possible to be previously undetected changes in the pathohistologically normal colon tissue in the proximity of the tumor.

Organoid Culture of Isolated Cells from Patient-derived Tissues with Colorectal Cancer.

Background:Colorectal cancer (CRC) is a heterogeneous disease; current research relies on cancer cell lines and animal cancer models, which may not precisely imitate inner human tumors and guide clinical medicine. The purpose of our study was to explore and further improve the process of producing three-dimensional (3D) organoid model and impel the development of personalized therapy.Methods:We subcutaneously injected surgically resected CRC tissues from a patient into BALB/c-nu mice to build patient-derived xenografts (PDXs). Isolated cells from PDXs at appropriate tumor size were mingled with Matrigel, and then seeded in ultra-low attachment 96-well plates at four cell densities (500, 1000, 2000, and 4000 single cells/well). Cells were cultured with advanced Dulbecco's Modified Eagle Medium/F12 medium additional with various factors added to maintain tumor's biological traits and growth activity. The growth curves of the four cell densities were measured after 24 h of culture until 25 days. We evaluated the effects of four chemotherapeutic agents on organoid model by the CellTiter-Glo® Luminescent Cell Viability Assay. Hematoxylin and eosin (H and E) staining of 3D organoids was performed and compared with patient and CRC PDX tissues. Furthermore, immunohistochemistry was performed, in which the organoids were stained with the proliferation marker, Ki-67. During the experimental process, a phase-contrast microscope was used.Results:Phenotype experimental results showed that 3D organoids were tightly packed together and grew robustly over time. All four densities of cells formed organoids while that composed of 2000 cells/well provided an adequate cultivation system and grew approximately 8-fold at the 25th day. The chemosensitivity of the four conventional drugs was [s]-10-hydroxycamptothecin > mitomycin C > adriamycin > paclitaxel, which can guide clinical treatment. Histological features of CRC patient's tumor tissues and mice tumor xenograft tissues were highly similar, with high-column-like epithelium and extracellular matrix. H and E-stained sections showed heterogeneous cell populations harbored in cancer organoids and were histologically similar to tumor tissues. The proliferation index was only 8.33% within spheroids, which exhibited confined proliferative cells that might be cancer stem cells.Conclusions:We successfully constructed a CRC organoid model that grew robustly over 25 days and demonstrated that 2000 cells/well in 96-well plate was a prime seeding density for cells to form organoids. The results confirmed that organoid model can be used for agent screening and personalized medicine.

Epigenetically regulated miR-145 suppresses colon cancer invasion and metastasis by targeting LASP1.

MiR-145 is a tumor-suppressive microRNA that participates in the malignant progression of colorectal cancer (CRC). Although miR-145 has been reported to inhibit proliferation and to induce apoptosis of CRC cells, the reports about its role in invasion and metastasis are controversial. The regulation of miR-145 its own expression also requires further elucidation. In this study, we firstly found that miR-145 is markedly downregulated in the metastatic tumors of CRC patients. Then through gain- and loss-of function studies, we demonstrated that miR-145 suppresses the invasion and metastasis of CRC cells. We also provided experimental evidences which include direct binding assays and verifications on tissue specimens to confirm that LIM and SH3 protein 1 (LASP1) is a direct target of miR-145. Furthermore, we identified the core promoter regions of miR-145 and observed the cooperation between histone methylation and transcription factors through binding to these core promoter regions to regulate the expression of miR-145 in CRC cells. Our study provides an insight into the regulatory network in CRC cells, thus offering new targets for treating CRC patients.

Abstract 2689: First MACC1 transgenic mice demonstrate tumor progression via the newly discovered MACC1/Nanog/Oct4 axis

Abstract We have previously identified the gene Metastasis-Associated in Colon Cancer 1 (MACC1). MACC1 acts a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Meanwhile, MACC1 has been established as a biomarker for tumor progression, metastasis and patient survival for a broad variety of solid cancer types. Here, we report for the first time the generation of transgenic mouse models for MACC1. We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with ApcMin mice (vil-MACC1/ApcMin). vil-MACC1/ApcMin mice significantly increased the total number of tumors (P = 0.0056). This was particularly apparent in large sized tumors (≥ 3 mm diameter; P = 0.0024). A detailed histopathological analysis of these lesions demonstrated that the tumors from the vil-MACC1/ApcMin mice had a more invasive phenotype together with an accelerated adenoma-carcinoma-sequence. Consequently, vil-MACC1/ApcMin mice showed a significantly reduced survival time as compared to ApcMin mice (P = 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/ApcMin mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors as compared to ApcMin controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro, and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r = 0.7005 and r = 0.6808, respectively; P &amp;gt; 0.0001 and P &amp;gt; 0.0002, respectively). Taken together, we provide first evidence that MACC1-induced tumor progression acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. The evidence is accumulated from experiments in cell culture, MACC1 transgenic animals as well as colorectal cancer patients. This is the first time that the function of MACC1 is linked to pathways crucial in cancer stem cells. Based on the hypothesis that cancer stem cells are the main engine behind tumor progression and metastasis, these findings might have important therapeutic implications. Specifically, MACC1's relevance as a biomarker might further be augmented by focusing on its expression in cancer stem cells. Furthermore, the connection of MACC1 with Oct4 and Nanog might offer novel options for therapeutic intervention to restrict tumor progression. Citation Format: Clara Lemos, Markus S. Hardt, Manisha Juneja, Cynthia Voss, Susann Förster, Boris Jerchow, Wolfram Haider, Hendrik Bläker, Ulrike S. Stein. First MACC1 transgenic mice demonstrate tumor progression via the newly discovered MACC1/Nanog/Oct4 axis. [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 2689.

Abstract 910: Genetic subclone heterogeneity of the human colon cancer initiating cell compartment

Abstract A subpopulation of tumor-initiating cells (TIC) drives colorectal cancer (CRC) progression in serial xenotransplantation. Strikingly, the CRC TIC compartment itself is heterogeneous and comprised of a hierarchy of long-term (LT-) TIC, tumor transient amplifying cells (T-TAC) and delayed contributing (DC-) TIC. Whether this functionally heterogeneous TIC compartment is genetically homogenous or whether distinct genetic subclones drive the functional heterogeneity of the TIC compartment is yet unknown. To address this question, we performed high coverage (91-126 fold) whole genome sequencing on primary CRC patient tumors (n = 3), corresponding serially passaged TIC enriched spheroids as well as spheroid derived serial xenografts. Sequenced samples harbored between 22.800 and 232.000 synonymous or non-synonymous single nucleotide variants (SNVs). In addition, all samples contained multiple focal or large-scale somatic copy number alterations (CNAs). Clustering of SNVs as well as subclonal copy numbers from serial xenografts and spheroids were used to define SNV- and CNA-based subpopulations. Next, cellular fractions of identified subpopulations were determined and combined applying maximal parsimony to create models of the minimal number of subclones present in each sample. Using this strategy, we found that multiple subclones were present in each sample analyzed. Subclone heterogeneity was maintained during serial in vitro passaging and serial xenografting. Importantly, tumor initiation in xenografts was driven by at least 3 distinct genetic subclones whose relative contribution dynamically changed over time. Strikingly, in serial samples from the same patients, different genetic subclones grew out in vivo and in vitro. To test whether functional heterogeneity of the TIC compartment is related to the presence of genetic subclones, we assessed the contribution of different TIC subtypes - LT-TIC, T-TAC and DC-TIC - at early and late time points of xenografting using secondary genetic marking. Therefore, 1×10⁁5 cells derived from early and late generation xenografts were transduced with an integrating lentiviral vector, thereby generating a stable barcode-like genetic mark which differs in each transduced cell. Following serial transplantation of transduced cells for 3 mouse generations, tumors were harvested and lentiviral integration sites were determined using highly sensitive LAM-PCR and high-throughput sequencing. Assessing the relative contribution of LT-TIC, T-TAC and DC-TIC revealed that the functional heterogeneity of the TIC compartment was preserved despite profound genetic subclone dynamics in serial xenotransplantation. These results strongly indicate that multiple genetic subclones drive long-term tumor formation and that functional heterogeneity of the CRC TIC compartment is not based on specific genetic subclones. Citation Format: Klara M. Giessler, Kortine Kleinheinz, Gnana Prakash Balasubramanian, Daniel Hübschmann, Taronish D. Dubash Rai, Sebastian D. Dieter, Christine Siegl, Christopher M. Hoffmann, Sarah Weber, Raffaele Fronza, Saira Afzal, Manfred Schmidt, Martin Schneider, Alexis Ulrich, Juergen Weitz, Wilko Weichert, Matthias Schlesner, Benedikt Brors, Claudia R. Ball, Hanno Glimm. Genetic subclone heterogeneity of the human colon cancer initiating cell compartment. [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 910.

MACC1 Induces Tumor Progression in Transgenic Mice and Colorectal Cancer Patients via Increased Pluripotency Markers Nanog and Oct4.

We have previously identified the gene MACC1 as a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Here, we report for the first time the generation of transgenic mouse models for MACC1. We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with Apc(Min) mice (vil-MACC1/Apc(Min)). vil-MACC1/Apc(Min) mice significantly increased the total number of tumors (P = 0.0056). This was particularly apparent in large tumors (≥3-mm diameter; P = 0.0024). A detailed histopathologic analysis of these lesions demonstrated that the tumors from the vil-MACC1/Apc(Min) mice had a more invasive phenotype and, consequently, showed a significantly reduced survival time than Apc(Min) mice (P = 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/Apc(Min) mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors compared with Apc(Min) controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r = 0.7005 and r = 0.6808, respectively; P > 0.0001 and P > 0.0002, respectively). We provide proof of principle that MACC1-induced tumor progression in colorectal cancer acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. These findings might have important implications for the design of novel therapeutic intervention strategies to restrict tumor progression. Clin Cancer Res; 22(11); 2812-24. ©2016 AACR.

Circulating and tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorectal cancer patients.

Mucosal associated invariant T (MAIT) cells are important for immune defense against infectious pathogens and regulate the pathogenesis of various inflammatory diseases. However, their roles in the development of colorectal cancer (CRC) are still unclear. This study examined the phenotype, distribution, clinical relevance and potential function of MAIT cells in CRC patients. We found that the percentages of circulating memory CD8+ MAIT cells were significantly reduced while tumor infiltrating MAIT cells were increased, especially in patients with advanced CRC. The serum CEA levels were positively correlated with the percentages of tumor infiltrating MAIT cells in CRC patients, but negatively correlated with the percentages of circulating MAIT in advanced CRC patients. Activated circulating MAIT cells from CRC patients produced lower IFN-γ, but higher IL-17. Furthermore, higher levels of Vα7.2-Jα33, IFN-γ and IL-17A were expressed in the CRC tissues. Co-culture of activated MAIT cells with HCT116 cells enhanced IL-17 expression and induced HCT116 cell cycle arrest at G2/M phase in a contact- and dose-dependent manner, which was abrogated by treatment with anti-MR1. Therefore, MAIT cells preferably infiltrate into the solid tumor in CRC patients and may participate in the immune surveillance of CRC.

Estrogen receptor β expression and colorectal cancer: a systematic review and meta-analysis.

Estrogen receptor β (ERβ) is a potential tumor-suppressor gene in colorectal cancer (CRC). This hypothesis is supported by clinical and laboratory observations. In this meta-analysis, we looked at studies that investigated the relationship between ERβ protein expression and CRC, comparing the lesion with normal adjacent mucosa. English medical literature searches were performed for ERβ expression in patients with CRC, tumor tissue versus normal mucosa. Searches were performed up to 31 May 2015, using MEDLINE, PubMed, EMBASE, Scopus, and CENTRAL. Meta-analysis was carried out using Comprehensive Meta-analysis Software. Pooled odds ratios and 95% confidence intervals were calculated and ERβ expression was compared in individual studies using the fixed-effects model. The odds ratio of ERβ expression was 0.216 (95% confidence interval 0.152-0.307, P<0.0001), lower in cancer tissue than normal mucosa. Funnel plot did not indicate a significant publication bias. There was no significant heterogeneity in the studies included: Q=5.897, d.f.(Q)=9, I=0.000, P=0.750. In this meta-analysis, we confirm the observation of decreased ERβ expression in CRC. Our results support the hypothesis of ERβ being a tumor-suppressor gene in the large bowel, and the ERβ protein protects against carcinogenesis and development of CRC when activated by estrogen. Further studies are needed to examine the potential of selective/specific ligands to activate ERβ without the side effects found with estrogen and without activating ERα. In this meta-analysis, we looked at studies that investigated the relationship between CRC and ERβ expression in the tumor and normal mucosa of CRC patients. English medical literature searches were performed for studies comparing ERβ expression in the cancer and normal colonic mucosa in patients with CRC. Meta-analysis was carried out, pooled odds ratios were calculated, and ERβ expression was compared in individual studies.

Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters.

Pleomorphic adenoma gene-like 1 gene (PLAGL1) encodes a zinc-finger nuclear transcription factor which promotes apoptosis and cell cycle arrest. Loss or downregulation of its expression has been observed in various human neoplasms. This study compared PLAGL1 expression in colorectal cancer (CRC) tissue and colon mucosa of healthy subjects at the mRNA and protein levels, and estimated its prognostic value. The PLAGL1 mRNA levels were also determined in CRC cell lines. We collected paired tumor tissue and unchanged mucosa of the large intestine from 121CRC patients as well as 72colon biopsies of healthy subjects obtained during screening colonoscopy. PLAGL1 mRNA levels were determined by quantitative PCR, while PLAGL1 protein expression was estimated by western blotting and immunohistochemistry. PLAGL1 mRNA level in tumor tissue was ~2-fold lower than in samples of corresponding unchanged tissues and biopsies of healthy colon mucosa. Downregulated expression of PLAGL1 mRNA was also observed in all tested CRC cell lines. Although the average content of PLAGL1 protein did not differ significantly between tumor and unchanged tissues of CRC patients or colon mucosa of healthy individuals, the decreased PLAGL1 protein levels in tumor specimens correlated with lymph node involvement, the presence of metastases and higher TNM disease stage. The PLAGL1 expression level did not correlate significantly with patient overall survival; however, the hazard ratio for patients whose tumor tissues showed reduced PLAGL1 immunohistochemical staining was twice higher than in patients with increased PLAGL1 immunoreactivity. In conclusion, these results suggest that dysregulation of PLAGL1 expression may be involved to some extent in the progression of CRC, but the so far collected patient survival data do not confirm applicability of the PLAGL1 expression level as a prognostic factor in CRC.

Glucose metabolism enzymes gene expression analysis and selective metabolic advantage in the progression of colorectal cancer (CRC).

e14519 Background: Cancer cells reprogram their metabolism to meet specific bioenergetic/biosynthetic needs. Abnormal expression of energy metabolism enzymes may sustain aggressive phenotypes, therapeutic resistance and it may disclose novel therapeutic targets. We analyzed mRNA expression of glucose metabolism, key-enzyme genes in normal mucosa (NM), primary tumor (PT) and liver metastasis (LM) of CRC patients (pts) who underwent surgery and systemic therapy for advanced disease. Methods: Tissues of chemotherapy-naive, non-diabetic CRC pts were retrospectively studied by RT-qPCR for mRNA expression of the following genes: hexokinase-1 (HK1) and 2 (HK2), embryonic pyruvate kinase (PKM2), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (SLC2A), voltage-dependent anion-selective channel protein-1 (VDAC1). The RT-qPCR ΔCt values (Cttarget–Ctreference) were used for calculating the expression level of each target gene with B2M and GUSB adopted as reference genes. A preliminary assessment was planned in...

Divergent expression patterns of SATB1 mRNA and SATB1 protein in colorectal cancer and normal tissues.

Special AT-rich sequence-binding protein 1 (SATB1) is a ‘genome organizer,’ and it has been proposed as a factor that affects the development and progression of various human neoplasms, including colorectal cancer (CRC). This study aimed to compare SATB1 expression in a group of CRC patients and healthy subjects at the mRNA and protein levels. We collected paired tumor tissue and unchanged mucosa of the large intestine from 102 CRC patients as well as 53 biopsies of normal colon mucosa obtained from healthy patients during screening colonoscopy. Tissue samples were quantified for SATB1 mRNA by quantitative PCR, while SATB1 protein expression was determined by Western blotting and immunohistochemistry. SATB1 mRNA level in tumor tissues was over twofolds lower than in samples of corresponding unchanged tissues and fourfolds lower than in biopsies of healthy colon mucosa. Western blotting analysis revealed that SATB1 protein content in tumor and unchanged tissues of CRC patients was over sixfold and fivefolds higher than in biopsies of healthy colon mucosa, respectively. Immunohistochemical staining demonstrated higher nuclear and cytoplasmic SATB1 reactivity in the tumor tissue compared to unchanged mucosa of CRC patients. Despite these differences, SATB1 mRNA, protein, and immunoreactivity levels did not correlate with patients’ clinicopathological data and their overall survival, but the latter analysis was limited by a relatively short period of follow-up. In conclusion, we suggest that some as yet unidentified posttranscriptional mechanisms that regulate SATB1 expression may be altered in the CRC tissue.

Evaluation of the left-to-right shift of colon tumors in Iran: Is the trend changing?

Background:Colorectal cancer (CRC) is the second cause of cancer-related deaths worldwide. There have been several studies reporting the proximal tumor shift, especially in Western countries. In the present study, we investigated the clinicopathologic and anatomical distributions of colorectal tumors in Iranian CRC patients.Materials and Methods:In this retrospective cohort study, 258 patients with CRC from 2008 to 2013 were evaluated. Comparison of variables was performed using Pearson's chi-square test and Fisher's exact test depending on the nature of the data.Results:A total of 258 patients including 124 (48.1%) females and 134 (51.9%) males enrolled in this study. The majority of cancers were detected in the rectosigmoid, i.e., 98 (38%) followed by the left colon, i.e., 84 (32.6%) and the right colon, i.e., 76 (29.5%). In the present study, we observed the significant association between metastases, adjuvant therapy, family history, and history of inflammatory bowel disease (IBD) with tumor, node, and metastasis (TNM) staging (P < 0.001). In univariate analysis, there was a strong association between overall survival (OS) and stage II CRC (P = 0.03). However, the predictive value was lost in multivariate analysis (P = 0.145).Conclusion:Unlike the majority of previous studies on Iranian CRC patients, we observed a considerably higher occurrence of right-sided colon cancers (84 versus 76). Although this phenomenon did not reach the statistical significance rate, based on recent studies on Iranian population including the present one, the pattern of anatomical distribution of colorectal tumors has been changed toward the proximal colon. This requires an urgent need to provide other strategies and complementary detecting approaches in order to identify proximal tumors in Iranian CRC patients.

Abstract 3894: Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101

Abstract BNC101 is a monoclonal antibody (mAb) targeting LGR5 currently undergoing IND-enabling studies in preparation for Phase I clinical studies in 2014. LGR5 is a validated cancer stem cell (CSC) receptor overexpressed in colorectal cancer (CRC), pancreatic cancer and most other solid tumors. Loss and gain-of-function studies indicate that LGR5 is a functional cancer receptor involved in the growth and survival of colon cancer cells in vitro and in vivo. Sorted LGR5+ primary CRC are highly tumorigenic compared to LGR5- cells in limiting dilution in vivo xenograft studies. Using Bionomics' CSC Rx Discovery™ platform, a panel of high affinity LGR5 mAbs were generated and screened for in vitro and in vivo activity against CSCs from patient-derived CRC tumors. LGR5 mAb therapeutic drug candidates were first screened and identified based on their ability to inhibit the formation of CRC CSC colonies in vitro. Top LGR5 mAb candidates were then tested as single agents and in combination with standard of care (SOC) chemotherapy in primary xenograft efficacy studies. Tumors from these studies were also harvested and re-implanted in limiting dilution assays (LDA) to assess in vivo anti-CSC activity of anti-LGR5 mAbs. A number of functional LGR5 mAbs with significant anti-tumor and anti-CSC activity were identified through this process. A lead BNC101 mAb clone was selected for clinical development based on its ability to inhibit CSC growth and key signaling pathways from multiple patient tumors both in vitro and in vivo. In LDA studies using two different CRC patient tumors, 5/8 (63%) and 6/8 (75%) mice implanted with serially diluted cells from BNC101 treated tumors remained tumor free, compared to 1/8 (13%) and 2/8 (25%) in the control groups. Combination with SOC chemotherapy was shown to further improve BNC101 activity in one CRC model, where 8/8 mice re-implanted for LDA remained tumor-free in a 6 month follow-up. BNC101 was also found to be highly active as a single agent and in combination with SOC in multiple pancreatic models and the MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model. In one primary pancreatic model, BNC101 combined with SOC completely eradicated 3/7 established tumors, compared to 0/7 tumors eradicated with control SOC alone. Additional translational data in CRC, pancreatic and TNBC supporting our hypothesis that BNC101 targeting of CSCs will significantly improve survival and increase duration of response in cancer patients will be presented. Citation Format: Peter Chu, Kristen Smith, John Norton, Colin Walsh, Farbod Shojaei, Jose Iglesias, Christopher Reyes. Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101. [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 3894. doi:10.1158/1538-7445.AM2014-3894

Abstract 5511: Direct effect of regorafenib on tyrosine kinase activities in treatment naive colorectal cancer (CRC) primary tumor tissue

Abstract Background: Regorafenib (BAY 73-4506) is the first small molecule tyrosine kinase inhibitor that has been approved for treatment of advanced CRC. In the Correct trial*, 1% of the participants showed a partial response and 42.8 % had stable disease. KRAS wild type and mutants showed equal response rates. Regorafenib is a multityrosine kinase inhibitor with nM IC50's against CRAF, VEGFR2, KIT and RET and is about tenfold less potent for BRAF, BRAFV600E, PDGFRβ and VEGFR1 and 3. We tested the effect of regorafenib and other angiogenic inhibitors on patient-derived kinases in treatment naive primary colon tumor samples for potency and selectivity of inhibition. Method: Colon tumor tissue from patients who had given informed consent was fresh frozen after surgical resection. All tumor specimens were analysed for BRAF and KRAS mutation status. Tissue cryosections were lysed in buffer with phosphatase and protease inhibitors. Phosphotyrosine kinase (PTK) and serine/threonine kinase (STK) activity profiles of tumour tissue were generated on PamChip® peptide microarrays comprising hundreds of peptide sequences derived from known human phosphorylation sites. The ex vivo effect of kinase inhibitors on these kinase activity profiles was determined. Peptide phosphorylation was monitored using fluorescently labelled antibodies. Data were analysed with Bionavigator software. Result: Kinase activity profiles were obtained from 16 CRC patient tumors for both tyrosine as well as serine/threonine specific peptide phosphorylations. Direct enzymatic inhibition of tumor-derived PTK activity was observed in all 16 samples at low micromolar concentrations (3 µM) of spiked-in regorafenib. For the serine/threonine kinase activity, spiking in of a high concentration of regorafenib (50 µM) resulted in only low levels of inhibition of phosphorylations on a subset of peptides, including BRAF substrate peptides. No difference in inhibition was observed between KRAS and BRAF mutants and wild type tumors. Furthermore the non-inhibited kinase activity profiles did not correlate to mutation status either. Conclusion: For the first time the direct biochemical effect of regorafenib has been tested on primary tumor tissue from CRC patients. These data support previous findings suggesting that regorafenib, besides its angiogenic activity on vasothelial cells, can inhibit oncogenic tyrosine kinases in the tumor. These results will be a basis for biomarker analysis by correlating on-chip drug effects to clinical responses. *Grothey et al, Lancet 2013 381(9863):303-312 Citation Format: Maria H. Hilhorst, Rosanne van den Oord, Hans Pruijt, Adriaan van den Brule, Peet Nooijen, Rik de Wijn, Liesbeth Houkes. Direct effect of regorafenib on tyrosine kinase activities in treatment naive colorectal cancer (CRC) primary tumor tissue. [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 5511. doi:10.1158/1538-7445.AM2014-5511

Plasma Bmi1 mRNA as a potential prognostic biomarker for distant metastasis in colorectal cancer patients.

Bmi1 is overexpressed in gastrointestinal cancers, including colorectal cancer (CRC); however, its role as a non-invasive biomarker in CRC has not been established. The aim of this study was to compare the plasma Bmi1 mRNA levels prior to and following curative resection of the primary tumor in CRC patients and to determine their association with the clinicopathological parameters. The plasma Bmi1 mRNA level was measured by quantitative polymerase chain reaction and expressed as cycle threshold value. There was no significant difference between the overall pre- and postoperative plasma Bmi1 mRNA level (31.73±2.63 vs. 31.93±2.88, respectively; P=0.614) in 45 CRC patients. However, when grouped into non-metastatic and metastatic CRC patients, the postoperative Bmi1 transcript level was found to be significantly lower compared to the preoperative level in patients with non-metastatic CRC (32.13±2.677 31.44±2.764, respectively; P=0.041), whereas there was a trend towards a higher postoperative Bmi1 transcript level compared to the preoperative level in the metastatic counterpart (30.85±3.916 vs. 33.27±0.718, respectively; P=0.164). Furthermore, when the patients were categorized into two groups according to their plasma Bmi1 postoperative vs. preoperative level status, we observed that patients without a reduction in the postoperative plasma Bmi1 mRNA levels exhibited a significantly higher rate of distant metastasis following primary resection (P=0.017) and a significantly worse prognosis regarding disease-free survival (P=0.016) when compared to the reduced postoperative plasma Bmi1 level counterparts. In conclusion, plasma Bmi1 mRNA levels may serve as a non-invasive biomarker for monitoring occult metastasis and predicting the development of distant metastasis.

Afatinib and its encapsulated polymeric micelles inhibits HER2-overexpressed colorectal tumor cell growth in vitro and in vivo.

Colorectal cancer (CRC) is known as a common malignant neoplasm worldwide. The role of EGFR/HER2 in CRC is unclear. Afatinib is an irreversible EGFR/HER2 inhibitor. There were few studies of afatinib on CRC. Here, we investigated the protein levels/expressions of HER2 in sera and tumors from CRC patients and the therapeutic effect of afatinib on HER2-overexpressed CRC in vitro and in vivo. The increased HER2 levels were detected in the collected sera and tumors of patients with CRC. The serological HER2 levels were correlated with the tumor HER2 expressions in patients. Afatinib also inhibited the HER2-positive tumor cell growth and caused apoptosis in HER2-overexpressed human colorectal cancer HCT-15 cells but not in low HER2 expressed human gastric cancer MKN45 cells. In vivo study showed that afatinib reduced tumor growth in HER2-overexpressed xenografts. Moreover, afatinib-encapsulated micelles displayed higher cytotoxic activity in HCT-15 cells and were more effective for tumor growth suppression in HCT-15-induced tumor xenografts than afatinib performance alone. Taken together, these findings suggest that higher serum HER2 levels reflect the higher HER2 contents in tumors of CRC patients, and the improved afatinib-encapsulated micelles possess high therapeutic efficacy in HER2-overexpressed CRC in vitro and in vivo.

HOTAIR long non-coding RNA is a negative prognostic factor not only in primary tumors, but also in the blood of colorectal cancer patients

Colorectal cancer (CRC) is one of the main causes of death of neoplasia. Demand for predictive and prognostic markers to reverse this trend is increasing. Long non-coding RNA HOTAIR (Homeobox Transcript Antisense Intergenic RNA) overexpression in tumors was previously associated with poor prognosis and higher mortality in different carcinomas. We analyzed HOTAIR expression levels in tumor and blood of incident sporadic CRC patients in relation to their overall survival with the aim to evaluate surrogate prognostic marker for CRC. Tissue donor group consisted of 73 CRC patients sampled for tumor and normal tissue. Blood donor group was represented by 84 CRC patients compared with 40 healthy controls. Patients were characterized for tumor-node-metastasis stage, tumor grade, microsatellite instability and tumor penetration by stromal cells. HOTAIR levels were assessed by real-time quantitative PCR. CRC patients had higher HOTAIR expression in blood than healthy controls (P = 0.0001), whereas there was no difference in HOTAIR levels between tumor and adjacent mucosa of CRC patients. HOTAIR levels positively correlated between blood and tumor (R = 0.43, P = 0.03). High HOTAIR levels in tumors were associated with higher mortality of patients [Cox's proportional hazard, hazard ratio = 4.4, 95% confidence interval: 1.0-19.2, P = 0.046]. The hazard ratio was even higher when blood HOTAIR levels were taken into account (hazard ratio = 5.9, 95% confidence interval: 1.3-26.1, P = 0.019). Upregulated HOTAIR relative expression in primary tumors and in blood of CRC patients is associated with unfavorable prognosis. Our data suggest that HOTAIR blood levels may serve as potential surrogate prognostic marker in sporadic CRC.

Significant association between IL23R and IL17F polymorphisms and clinical features of colorectal cancer

Th17cells are involved in inflammatory and autoimmune diseases. These cells may be involved in pathological processes mainly producing pro-inflammatory cytokines. Recently, it was shown that the IL23/IL17 pathway plays an important role in the development of inflammatory bowel disease. In general, genes encoding cytokines are genetically polymorphic and polymorphisms in genes IL23R el IL17F were shown associated with susceptibility to Crohn's disease and ulcerative colitis which in their turn are considered as risk factors for developing colorectal cancer (CRC). Our approach is to study IL17F and IL23R polymorphisms as risk factor associated with CRC in the Tunisian population in patients and healthy controls. Interesting, we noted a significant association between IL17F and IL23R polymorphisms and tumor location (p=0.0001 and p=0.049, respectively), tumor histology (p=0.007 and p=0.049, respectively) and tumor architecture (p=0.0000000001 and p=0.07, respectively) in CRC patients. We also showed a significant association of IL17F variant with an increased risk of TNM stage III/IV (p=0.007), showing an increased risk of advanced stage. Finally, we observed a positive link between IL17F polymorphism and CRC patients with lymph nodes (p=0.0000000001) and metastasis (p=0.00000009). However, we found no evidence to support a significant association between IL17F and IL23R polymorphisms and colorectal cancer susceptibility. Our findings suggest that IL17F and IL23R polymorphisms were significantly associated with clinical features variables. The IL17F cytokine appear to be involved in the control of tumor growth and invasion of gastrointestinal tumors. IL17 and IL23 polymorphisms or those of their receptors as important determinants of susceptibility to colorectal cancer are still subject to questioning.

Abstract A48: Therapeutic targeting of colorectal cancer stem cells with BNC101, a functional anti-LGR5 monoclonal antibody.

Abstract BNC101 is a monoclonal antibody that targets LGR5, a validated cancer stem cell (CSC) receptor overexpressed in colorectal cancer (CRC) and most other solid tumors. Loss and gain-of-function studies found that LGR5 is a functional cancer receptor involved in the growth and survival of colon cancer cells in vitro and in vivo. Sorted LGR5+ primary CRC cells were highly tumorigenic compared to LGR5- cells in limiting dilution in vivo xenograft studies. Using Bionomics’ CSC Rx Discovery™ platform, a panel of high affinity LGR5 mAbs was generated and screened for in vitro and in vivo activity against CSCs from patient-derived CRC tumors. In vitro assays initially identified top LGR5 mAb drug candidates based on their ability to inhibit the formation of CSC colonies. In vivo assays tested candidate LGR5 mAbs as single agents and in combination with standard of care (SOC) chemotherapy in primary xenograft efficacy studies. Tumors from these studies were then harvested and re-implanted in limiting dilution assays (LDA) to assess anti-CSC activity. A number of functional LGR5 mAbs with significant anti-tumor and anti-CSC activity were identified through this process. The lead BNC101 mAb clone was selected for clinical development based on its ability to inhibit CSC growth and key signaling pathways from multiple patient tumors both in vitro and in vivo. In LDA studies using two different CRC patient tumors, 5/8 (63%) and 6/8 (75%) mice implanted with serially transplanted cells from BNC101 treated tumors remained tumor free, compared to 1/8 (13%) and 2/8 (25%) in the control groups. Combination with SOC chemotherapy was shown to further improve BNC101 activity in one CRC model, where 8/8 mice re-implanted for LDA remained tumor-free in a 6 month follow-up. BNC101 was also found to be active against tumors from additional cancer indications outside of CRC. Current research is focused on advancing BNC101 translational studies to support clinical development. Significant progress and new results in these studies will be presented, in particular the identification of optimal BNC101 chemo combination strategies, biomarkers and mechanism of action. Altogether, the data support our therapeutic hypothesis that anti-LGR5 mAb BNC101 treatment will be part of an effective strategy in combination with SOC therapy to significantly improve survival and increase duration of response for patients with colorectal cancer and other solid tumors. BNC101 is currently undergoing IND-enabling manufacturing and safety studies in preparation for Phase I clinical studies scheduled for 2014. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A48. Citation Format: Peter Chu, Kristen Smith, Farbod Shojaei, Colin Walsh, John Norton, Jose Iglesias, Christopher Reyes. Therapeutic targeting of colorectal cancer stem cells with BNC101, a functional anti-LGR5 monoclonal antibody. [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 A48.

Abstract B245: Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody.

Abstract In metastatic colorectal cancer (CRC) patients, chemotherapy in combination with targeted therapies such as Avastin, Erbitux, and Vectibix (monoclonal antibodies) has improved response rates with prolongation of progression free survival and overall survival. Despite these major therapeutic progresses, at least 30 to 50% of patients remain non-responders. Our project aimed to identify and validate new molecular targets of CRC. Comparison of gene expression profiles between normal mucosa and primary tumor of CRC patients followed by immunohistochemistry analysis identified claudin-1 (CLDN1) as potential therapeutic target for antibody-targeted therapy. CLDN1 is a major tight junction transmembrane protein, already found overexpressed in CRC in several studies and associated with tumor progression. We have generated monoclonal antibodies (mAb) directed against extracellular domains of CLDN1 and demonstrated their specificity against human CLDN1 in vitro and in vivo. Then, using mice xenografted with the CRC cell line SW620 we tested the therapeutic effect of the 6F6C3mAb. The mice were treated by i.p. injections with vehicle or 6F6C3mAb at two different doses. The results showed that 6F6C3mAb treated-groups had a significant (p= 0,018) reduced growth compared to the control group and this effect was dose-dependent. The in vitro effects of the binding of 6F6C3mAb to CLDN1 were also assessed by clonogenic assays. Treatment by 6F6C3mAb induced a significant (p&amp;lt; 0.05) reduction of the number of colony-forming cells for CLDN1high colorectal cancer cells (SW620, Caco2) but also for CLDN1high ovarian and pancreatic cancer cells (PANC1, BXPC3, SKOV3, IGROV1). Thus, 6F6C3mAb binding had a profound effect on cancer cell survival. In conclusion, CLDN1 seems to be a promising target for antibody-based therapy. The 6F6C3mAb is able to inhibit tumor growth and tumor survival by binding the extracellular domains of CLDN1. It can be used in the treatment of cancer diseases such as colorectal cancer as well in adjuvant as in metastatic setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B245. Citation Format: Adeline Ayrolles-Torro, Nadia Vezzio-Vie, Vincent Denis, Florence Boissiere-Michot, Véronique Garambois, Muriel Busson, Imade Ait Arsa, Caroline Mollevi, Martine Pugniere, Frédéric Bibeau, Pierre Martineau, Céline Gongora, Maguy Del Rio. Claudin-1 (CLDN1) as a new therapeutic target in colorectal cancer: Inhibition of cell growth and survival by an anti-CLDN1 monoclonal antibody. [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 B245.