Lindsay Bennett

NFκB signalling is upregulated in a subset of castrate-resistant prostate cancer patients and correlates with disease progression.

Background:Cell line models suggest that activation of NFκB is associated with progression of prostate cancer. This pathway may be a therapeutic target if these observations translate to clinical specimens.Methods:Immunohistochemistry measured NFκBp65 (p65), NFκBp65 nuclear localisation signal (NLS), NFκBp65 phosphorylated at ser 276 (p65ser276), NFκBp65 phosphorylated at ser 536 (p65ser536), IκBα phosphorylated at ser 32/36 (pIκBαser32/36) and MMP-9 protein expression in 61 matched hormone naive prostate cancer (HNPC) and castrate-resistant prostate cancer (CRPC) tumours. Animal and cell models were used to investigate the role of NFκB inhibition in prostate carcinogenesis.Results:In HNPC tumours, NLS expression significantly associated with a shorter time to disease recurrence and disease-specific death. In CRPC tumours p65, pIκBαser32/36 and MMP-9 expression significantly associated with shorter time to death from disease recurrence and shorter disease-specific death. MMP-9 and pIκBαser32/36 expression significantly associated with metastases at recurrence and were independent of Gleason sum and prostate-specific antigen at recurrence. Expression of phosphorylated Akt was associated with increased p65 activation in mouse models and inhibition of NFκB in LNCaP cells significantly reduced cellular proliferation and induced apoptosis.Conclusion:These results provide further evidence that the NFκB pathway could be exploited as a target for CRPC.

IGFBP-5 enhances epithelial cell adhesion and protects epithelial cells from TGFβ1-induced mesenchymal invasion

TGFβ1 is a major fibrotic factor and its actions involve induction of epithelial cell death, together with the stimulation and transdifferentiation of fibroblasts into collagen- and fibronectin-secreting myofibroblasts. These actions of TGFβ1 are also consistent with a pro-metastatic role, by aiding epithelial cell escape through mesenchymal tissues. Recently IGFBP-5 has been described as a pro-fibrotic (pro-metastatic?) agent and the aim of this study was to compare and contrast the actions of IGFBP-5 with TGFβ1. We used NMuMG cells and cloned stable epithelial and mesenchymal lines from the parent cells. TGFβ1 induced apoptosis and/or EMT in the epithelial cells, whereas it enhanced mesenchymal cell survival and migration. IGFBP-5, in contrast, enhanced both cell-cell and cell-ECM adhesion and also improved wound closure in epithelial cells whereas, in mesenchymal cells, IGFBP-5 decreased adhesion and migration. Furthermore, IGFBP-5 was able to antagonise the actions of TGFβ1. In a co-culture model simulating epithelial-mesenchymal boundaries, IGFBP-5 was able to antagonise the disruptive transgressions induced by TGFβ1. Overall, these findings suggest that IGFBP-5 is important in maintaining epithelial-mesenchymal boundaries and thus may limit metastasis and fibrosis by inducing an orderly repair mechanism, very distinct from the fibrotic disruption induced by TGFβ1. A role for IGFBP-5 in the inhibition of metastasis is supported by immunohistochemical studies of breast cancer microarrays, where we show that elevated IGFBP-5 expression is associated with increased disease-free survival.

Androgen Receptor Phosphorylation at Serine 308 and Serine 791 Predicts Enhanced Survival in Castrate Resistant Prostate Cancer Patients

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR94), 308 (pAR308), 650(pAR650) and 791 (pAR791). No correlations with clinical parameters were observed for pAR94 or pAR650 in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR308 is significantly associated with a longer time to disease specific death (p = 0.011) and high pAR791 expression significantly associated with a longer time to disease recurrence (p = 0.018) in HNPC tumours and longer time to death from disease recurrence (p = 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p = 0.022) and low proliferating tumours (p = 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer.

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)-positive breast cancer

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico‐pathological characteristics. After siRNA‐mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA‐MB‐231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease‐free survival (p = 0.029) and recurrence‐free survival on tamoxifen (p < 0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer‐specific survival (p = 0.012 and p = 0.007, respectively) and recurrence‐free survival on tamoxifen (p = 0.013 and p < 0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer‐specific survival (p = 0.023), disease‐free survival (p = 0.002) and recurrence‐free survival on tamoxifen (p = 0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non‐canonical NF‐κB pathway was inducible by exposure to lymphotoxin in ER‐positive MCF7 cells and not in ER‐negative MDA‐MB‐231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA‐MB‐231 cells. IKKα is an important determinant of poor outcome in patients with ER‐positive invasive ductal breast cancer and thus may represent a potential therapeutic target.

The relationship between members of the canonical NF-κB pathway, components of tumour microenvironment and survival in patients with invasive ductal breast cancer

The aim of the present study was to examine the relationship between tumour NF-κB activation, tumour microenvironment including local inflammatory response (LIR) and cancer-specific survival in patients with operable ductal breast cancer. Immunohistochemistry (tissue microarray of 376 patients) and western blotting (MCF7 and MDA-MB-231 breast cancer cells) was performed to assess expression of key members of the canonical NF-κB pathway (inhibitory kappa B kinase (IKKβ) and phosphorylated p65 Ser-536 (p-p65)). Following silencing of IKKβ, cell viability and apoptosis was assessed in both MCF7 and MDA-MB-231 cell lines. P-p65 was associated with cancer-specific survival (CSS) (nuclear P=0.042 and total P=0.025). High total p-p65 was associated with increase grade tumour grade (P=0.010), ER positivity (P=0.023), molecular subtype (P=0.005), lower Klintrup-Makinen grade (P=0.013) and decreased CD138 count (P=0.032). On multivariate analysis, total p-p65 expression independently associated with poorer CSS (P=0.020). In vitro work demonstrated that the canonical NF-κB pathway was inducible by exposure to TNFα in ER-positive MCF7 cells and to a lesser extent in ER-negative MDA-MB-231 cells. Reduction of IKKβ expression by siRNA transfection increased levels of apoptosis and reduced cell viability in both MCF7 (P=<0.001, P=<0.001, respectively) and MDA-MB-231 cells (P=>0.001, P=0.002, respectively). This is consistent with the hypothesis that canonical IKKβ-NF-κB signalling drives tumour survival. These results suggest that activation of the canonical NF-κB pathway is an important determinant of poor outcome in patients with invasive ductal breast cancer.

Relationship between tumour PTEN/Akt/COX-2 expression, inflammatory response and survival in patients with colorectal cancer

In patients with colorectal cancer (CRC), local and systemic inflammatory responses have been extensively reported to associate with cancer survival. However, the specific signalling pathways responsible for inflammatory responses are not clear. The PTEN/Akt pathway is a plausible candidate as it may play a role in mediating inflammation via COX-2, and has been associated with cancer progression. This study therefore examined the relationship between tumour PTEN/Akt/COX-2 expression, inflammatory responses and survival in CRC patients using a tissue microarray. In 201 CRC patients, activation of tumour-specific PTEN/Akt significantly associated with poorer CSS (12.0yrs v 7.3yrs, P=0.032), poorer differentiation (P=0.032), venous invasion (P=0.008) and peritoneal involvement (P=0.004). Patients were stratified for peri-nuclear expression of COX-2 to examine associations with inflammatory responses. In patients with absent peri-nuclear COX-2 expression, activation of tumour-specific PTEN/Akt significantly associated with poorer CSS (11.9yrs v 5.4yrs, P=0.001), poorer differentiation (P=0.018), venous invasion (P=0.003) and peritoneal involvement (P=0.001). However, no associations were seen with either the local or systemic inflammatory responses. In CRC patients, tumour-specific PTEN/Akt pathway activation was significantly associated with poorer CSS, particularly when peri-nuclear COX-2 expression was absent. However, activation of the PTEN/Akt pathway appears not to be responsible for the regulation of inflammatory responses.

Predictive biomarkers for endocrine therapy: retrospective study in Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial

Background Aromatase inhibitors improve disease-free survival compared with tamoxifen in postmenopausal women with hormone receptor-positive breast cancer. The Tamoxifen and Exemestane Adjuvant Multinational (TEAM) trial compared exemestane monotherapy with sequential therapy of tamoxifen followed by exemestane. The trial failed to show a statistically significant difference between treatment arms. A robust translational program was established to investigate predictive biomarkers. Methods A tissue microarray was retrospectively constructed using a subset of patient tissues (n = 4631) from the TEAM trial (n = 9766). Immunohistochemistry was performed for biomarkers, classed into three groups: MAPK pathway, NF-kappa B pathway, and estrogen receptor (ER) phosphorylation. Expression was analyzed for association with relapse-free survival (RFS) at 2.5 and 10 years and treatment regimen using Kaplan-Meier curves and log-rank analysis. All statistical tests were two-sided. Results In univariate analysis, ER167 (hazard ratio [HR] = 0.71, 95% confidence interval [CI] = 0.59 to 0.85, P < .001), IKKα (HR = 0.74, 95% CI = 0.60 to 0.92, P = .005), Raf-1338 (HR = 0.64, 95% CI = 0.52 to 0.80, P < .001), and p44/42 MAPK202/204 (HR = 0.77, 95% CI = 0.64 to 0.92, P = .004) were statistically significantly associated with improved RFS at 10 years in patients receiving sequential therapy. Associations were strengthened when IKKα, Raf-1338, and ER167 were combined into a cumulative prognostic score (HR = 0.64, 95% CI = 0.52 to 0.77, P < .001). Patients with an all negative IKKα, Raf-1338, and ER167 score favored exemestane monotherapy (odds ratio = 0.56, 95% CI = 0.35 to 0.90). In multivariable analysis, the IKKα, Raf-1338, and ER167 score (P = .001) was an independent prognostic factor for RFS at 10 years in patients receiving sequential therapy. Conclusions The IKKα, Raf-1338, and ER167 score is an independent predictive biomarker for lower recurrence on sequential therapy. Negative expression may further offer predictive value for exemestane monotherapy.

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

A novel tumor-based epithelial-to-mesenchymal transition score that associates with prognosis and metastasis in patients with stage II/III colorectal cancer: Tumour EMT as a predictive marker for micrometastasis in colorectal cancer

It is increasingly appreciated that host factors within the tumor center and microenvironment play a key role in dictating colorectal cancer (CRC) outcomes. As a result, the metastatic process has now been defined as a result of epithelial–mesenchymal transition (EMT). Establishment of the role of EMT within the tumor center and its effect on the tumor microenvironment would be beneficial for prognosis and therapeutic intervention in CRC. The present study assessed five immunohistochemical EMT markers within the tumor center on a 185 Stage II/III CRC patient tissue microarray. In 185 patients with CRC, cytoplasmic snail (HR 1.94 95% confidence interval [CI] 1.15–3.29, p = 0.012) and a novel combined EMT score (HR 3.86 95% CI 2.17–6.86, p < 0.001) were associated with decreased cancer‐specific survival. The combined EMT score was also associated with increased tumor budding (p = 0.046), and systemic inflammation (p = 0.007), as well as decreased memory T‐cells within the stroma (p = 0.030) and at the invasive margin (p = 0.035). Furthermore, the combined EMT score was associated with cancer‐specific survival independent of TNM‐stage (HR 4.12 95% CI 2.30–7.39, p < 0.001). In conclusion, a novel combined EMT score stratifies patients survival in Stage II/III CRC and associates with key factors of tumor metastasis. Therefore, the combined EMT score could be used to identify patients at risk of micrometastases and who may benefit from standard adjuvant therapy, potentially in combination with EMT blockade.

Abstract B125: The relationship between members of the canonical NF-κB pathway, components of the microenvironment and survival in patients with colorectal cancer

The canonical NF-κB pathway regulates transcription of a wide range of genes involved in various processes including inflammation and proliferation, suggesting that it might provide a link between the systemic inflammatory response (SIR), local inflammatory response (LIR) and tumor signalling, the present study was to examine the relationship between SIR, tumor NF-κB pathway, tumor microenvironment including LIR and cancer specific survival in patients with colorectal cancer Immunohistochemistry of inhibitory kappa β kinase (IKK)-β, p65 (Rel A) and phosphorylated p65 (p-p65) was performed using colorectal cancer tissue microarray of 151 patients and the weight histoscore method was employed to assess protein expression. The relationship between these proteins, BRAF status, tumor stroma percentage, local inflammation, systemic inflammation, and cancer-specific survival were examined. In addition, western blotting was performed to assess expression of key members of the canonical NF-κB pathway following stimulation with either TNFα or IL-1 in BRAF wildtype (T84) and BRAF V600E mutated cells (HT-29). Expression of neither p65 nor p-p65, at any cellular location, was associated with clinical outcome measures. However a significant association between cancer specific survival and cytoplasmic IKKβ (p = 0.015), was observed and 10 year survival stratified from 78% to 59%. In addition, when patients were stratified by BRAF status the association with cancer specific survival and cytoplasmic IKKβ was upheld in those patients with wild type BRAF (p = 0.014) but negated in those with mutated BRAF (p = 0.287). Cytoplasmic IKKβ was significantly associated with venous invasion (p = 0.024), immunoscore (p = 0.023), Klintrup-Makinen grade (p = 0.050) and Glasgow microenvironment score. Total cell IKKβ expression was also associated with cancer specific survival, (p = 0.015) and 10 year survival was stratified from 73% to 53%. Total cell IKKβ was significantly associated with TNM (p = 0.004), tumor differentiation (p = 0.001), venous invasion (p = 0.011), necrosis (p = 0.003) and tumor stoma percentage (p = 0.048). In vitro, the canonical NF-κB pathway was induced in colon cancer cell lines T84 and HT-29 by exposure to either TNFα or IL-1 (p-p65 levels increased at 15 min), IKKβ expression remained constant. In colorectal cancer patients, cytoplasmic and total cell IKKβ expression is associated with cancer specific survival and this was related to tumor microenviroment and LIR but not the SIR. Interactions between stromal and NF-κB pathway may be an important method by which disease progression occurs. Citation Format: Jean A. Quinn, Lindsay Bennett, Antonia Roseweir, James H. Park, Paul G. Horgan, Donald C. McMillan, Joanne Edwards. The relationship between members of the canonical NF-κB pathway, components of the microenvironment and survival in patients with colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B125.