Michelle Thomas

STAT1 A Modulator of Chemotherapy-induced Apoptosis

The anthracyclines, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that these drugs could activate the transcription factor, nuclear factor κB, in a DNA damage-dependent manner. We now show that these drugs can potentiate the activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 breast cancer cells treated with IFN-γ. We observed that key markers of STAT1 activation, including tyrosine 701 and serine 727 phosphorylation, were enhanced in the presence of doxorubicin. This potentiation resulted in enhanced nuclear localization of activated STAT1 and led to an increase in the nuclear binding of activated STAT complexes. The observed potentiation was specific for STAT1 and IFN-γ, as no effects were observed with either STAT3 or STAT5. Furthermore, the type I IFNs (α and β) had little or no effect. The observed effects on STAT1 phosphorylation have previously been linked with maximal transcriptional activation and apoptosis. Cell viability was assessed by crystal violet staining followed by analysis with CalcuSyn to determine combination index values, a measure of synergy. We confirmed that significant synergy existed between IFN-γ and doxorubicin (combination index = 0.34) at doses lower than IC50 values for this drug (0.67 μmol/L). In support of this, we observed that apoptotic cell death was also enhanced by measuring poly(ADP-ribose) polymerase and caspase-3 cleavage. Finally, suppression of STAT1 expression by small-interfering RNA resulted in a loss of synergistic apoptotic cell death compared with cells, where no suppression of STAT1 expression was attained with scrambled small-interfering RNA control. We conclude that doxorubicin potentiates STAT1 activation in response to IFN-γ, and that this combination results in enhanced apoptosis in breast cancer cells.The anthracyclines, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that these drugs could activate the transcription factor, nuclear factor kappaB, in a DNA damage-dependent manner. We now show that these drugs can potentiate the activation of signal transducer and activator of transcription 1 (STAT1) in MDA-MB 435 breast cancer cells treated with IFN-gamma. We observed that key markers of STAT1 activation, including tyrosine 701 and serine 727 phosphorylation, were enhanced in the presence of doxorubicin. This potentiation resulted in enhanced nuclear localization of activated STAT1 and led to an increase in the nuclear binding of activated STAT complexes. The observed potentiation was specific for STAT1 and IFN-gamma, as no effects were observed with either STAT3 or STAT5. Furthermore, the type I IFNs (alpha and beta) had little or no effect. The observed effects on STAT1 phosphorylation have previously been linked with maximal transcriptional activation and apoptosis. Cell viability was assessed by crystal violet staining followed by analysis with CalcuSyn to determine combination index values, a measure of synergy. We confirmed that significant synergy existed between IFN-gamma and doxorubicin (combination index = 0.34) at doses lower than IC(50) values for this drug (0.67 micromol/L). In support of this, we observed that apoptotic cell death was also enhanced by measuring poly(ADP-ribose) polymerase and caspase-3 cleavage. Finally, suppression of STAT1 expression by small-interfering RNA resulted in a loss of synergistic apoptotic cell death compared with cells, where no suppression of STAT1 expression was attained with scrambled small-interfering RNA control. We conclude that doxorubicin potentiates STAT1 activation in response to IFN-gamma, and that this combination results in enhanced apoptosis in breast cancer cells.

Cellular FLICE-inhibitory protein regulates chemotherapy- induced apoptosis in breast cancer cells

Combination treatment regimens that include topoisomerase-II–targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we showed that IFN-γ and doxorubicin cotreatment synergistically induced apoptosis in MDA435 breast cancer cells in a signal transducer and activator of transcription 1–dependent manner. In this study, we found that this synergy was caspase-8 dependent. In addition, we found that IFN-γ down-regulated the expression of the caspase-8 inhibitor cellular FLICE-inhibitory protein (c-FLIP). Furthermore, IFN-γ down-regulated c-FLIP in a manner that was dependent on the transcription factors signal transducer and activator of transcription 1 and IFN regulatory factor-1. However, IFN-γ had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a posttranscriptional level following IFN-γ treatment. Characterization of the functional significance of c-FLIP modulation by small interfering RNA gene silencing and stable overexpression studies revealed it to be a key regulator of IFN-γ– and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-γ–induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitized MDA435 cells to other chemotherapies, including etoposide, mitoxantrone, and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells. [Mol Cancer Ther 2007;6(5):1544–51]

Over-expression of cathepsin E and trefoil factor 1 in sessile serrated adenomas of the colorectum identified by gene expression analysis

Sessile serrated adenomas are now recognised as precursor lesions of a substantial subset of colorectal cancers arising via a so-called “serrated pathway”. However, their biological markers remain to be defined. The aim of our study was to identify differentially expressed genes in sessile serrated adenomas and conventional adenomas. Gene expression analysis demonstrated molecular differences between polyp types. Further studies using quantitative real-time polymerase chain reaction on cathepsin E (CTSE) demonstrated a significantly (p < 0.05) higher expression in sessile serrated adenomas as compared to hyperplastic polyp and tubular adenomas. Trefoil Factor 1 showed the same trend of expression for sessile serrated adenomas as compared to hyperplastic polyps and was significantly higher in both polyps compared to tubular adenomas. Immunohistochemistry for both proteins demonstrated strong cytoplasmic staining of abnormal crypts in all sessile serrated adenomas, while staining in tubular adenomas and hyperplastic polyps was absent or weak and focal. BRAF and KRAS mutation analysis were employed to further validate polyp discrimination. The findings demonstrated the positive association of the BRAF mutation, V600E, with sessile serrated adenomas and KRAS mutations with tubular adenomas (p < 0.05). This study demonstrates the over-expression in CTSE, in particular, and TFF1 in sessile serrated adenomas compared to both hyperplastic polyps and tubular adenomas.

Argon Plasma Coagulation Therapy Versus Topical Formalin for Intractable Rectal Bleeding and Anorectal Dysfunction After Radiation Therapy for Prostate Carcinoma

PURPOSE To evaluate and compare the effect of argon plasma coagulation (APC) and topical formalin for intractable rectal bleeding and anorectal dysfunction associated with chronic radiation proctitis. METHODS AND MATERIALS Thirty men (median age, 72 years; range, 49-87 years) with intractable rectal bleeding (defined as ≥1× per week and/or requiring blood transfusions) after radiation therapy for prostate carcinoma were randomized to treatment with APC (n=17) or topical formalin (n=13). Each patient underwent evaluations of (1) anorectal symptoms (validated questionnaires, including modified Late Effects in Normal Tissues-Subjective, Objective, Management, and Analytic and visual analogue scales for rectal bleeding); (2) anorectal motor and sensory function (manometry and graded rectal balloon distension); and (3) anal sphincteric morphology (endoanal ultrasound) before and after the treatment endpoint (defined as reduction in rectal bleeding to 1× per month or better, reduction in visual analogue scales to ≤25 mm, and no longer needing blood transfusions). RESULTS The treatment endpoint was achieved in 94% of the APC group and 100% of the topical formalin group after a median (range) of 2 (1-5) sessions of either treatment. After a follow-up duration of 111 (29-170) months, only 1 patient in each group needed further treatment. Reductions in rectal compliance and volumes of sensory perception occurred after APC, but no effect on anorectal symptoms other than rectal bleeding was observed. There were no differences between APC and topical formalin for anorectal symptoms and function, nor for anal sphincteric morphology. CONCLUSIONS Argon plasma coagulation and topical formalin had comparable efficacy in the durable control of rectal bleeding associated with chronic radiation proctitis but had no beneficial effect on anorectal dysfunction.

Claudin-1 Expression Is Elevated in Colorectal Cancer Precursor Lesions Harboring the BRAF V600E Mutation.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/P) are now recognised precursors of colorectal cancer (CRC) including cancers harbouring somatic BRAF (V600E) mutations. While the morphological diagnostic criteria of SSA/P have been established, distinguishing between small/early SSA/P and microvesicular hyperplastic polyps (MVHP) is challenging and may not be possible in routine practice. METHODS: Gene expression profiling of MVHP (n=5, all BRAF V600E wild-type) and SSA/P (n=5, all BRAF V600E mutant) samples was performed. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and immunohistochemical analysis was performed to verify the expression of claudin 1 (CLDN1) in MVHP and SSA/P. RESULTS: Gene expression profiling studies conducted between MVHP and SSA/P identified CLDN1 as the most statistically significant differentially expressed gene (p<0.05). Validation with qRT-PCR confirmed an up-regulation of CLDN1 in BRAF V600E mutant polyps regardless of polyp type (p<0.0005). Immunohistochemical analysis of CLDN1 expression in BRAF V600E mutant SSA/Ps (n=53) and MVHPs (n=111) and BRAF wild-type MVHPs (n=58), demonstrated a strong correlation between CLDN1 expression and the BRAF V600E mutation in both SSA/P and MVHP samples when compared to wild-type polyps (p<0.0001). CONCLUSION: This study demonstrates an up regulation of CLDN1 protein in serrated colorectal polyps including MVHP harbouring the BRAF V600E mutation. Our results demonstrated an apparent heterogeneity on the molecular level within the MVHP group and suggest that MVHP with somatic BRAF V600E mutation and up-regulated expression of CLDN1 are closely related to SSA/P and may in fact represent a continuous spectrum of the same neoplastic process within the serrated pathway of colorectal carcinogenesis.

Conventional adverse features do not predict response to adjuvant chemotherapy in stage II colon cancer.

The role of adjuvant chemotherapy in patients with stage II colon cancer is unclear. Current guidelines recommend adjuvant chemotherapy for high‐risk patients, although the benefit demonstrated to date is small. Our study examined if adjuvant chemotherapy is associated with improved cancer‐specific survival in high‐risk patients with stage II colon cancer.

Is extended thromboprophylaxis necessary in elective colorectal cancer surgery

Colorectal cancer surgery carries a high risk of venous thromboembolism (VTE) but the optimal duration of thromboprophylaxis is unknown. The cost‐effectiveness of extended prophylaxis is not known in Australasia. The aims of this study were to determine the 30‐day incidence of VTE in patients undergoing colorectal cancer surgery, to audit compliance with thromboprophylaxis protocols and to estimate the cost of treating all patients for 28 days with enoxaparin.

Clinicopathological predictors of benefit from adjuvant chemotherapy for stage C colorectal cancer: Microsatellite unstable cases benefit.

In colorectal cancer (CRC), adjuvant therapy is offered on the basis of stage and attempts to identify factors to better target treatment have not been successful. Recent work suggested that mismatch repair deficient CRCs may not benefit from 5FU adjuvant chemotherapy but studies remain conflicting. We aimed to determine if gender, tumor site, tumor pathological characteristics and microsatellite instability (MSI) predict survival benefit from adjuvant chemotherapy in stage C CRC.

Early surgery in Crohn’s disease a benefit in selected cases

AIM To compare the outcomes of a cohort of Crohns disease (CD) patients undergoing early surgery (ES) to those undergoing initial medical therapy (IMT). METHODS We performed a review of a prospective database CD patients managed at a single tertiary institution. Inclusion criteria were all patients with ileal or ileocolonic CD between 1995-2014. Patients with incomplete data, isolated colonic or perianal CD were excluded. Primary endpoints included the need for, and time to subsequent surgery. Secondary endpoints included the number and duration of hospital admissions, and medical therapy. RESULTS Forty-two patients underwent ES and 115 underwent IMT. The operative intervention rate at 5 years in the ES group was 14.2% vs IMT 31.3% (HR = 0.41, 95%CI: 0.23-0.72, P = 0.041). The ES group had fewer hospital admissions per patient [median 1 vs 3 (P = 0.012)] and fewer patients required anti-TNF therapy than IMT (33.3% vs 57%, P = 0.003). A subgroup analysis of 62 IMT patients who had undergone surgery were compared to ES patients, and showed similar 5 year (from index surgery) re-operation rates 16.1% vs 14.3%. In this subset, a significant difference was still found in median number of hospital admissions favouring ES, 1 vs 2 (P = 0.002). CONCLUSION Our data supports other recent studies suggesting that patients with ileocolonic CD may have a more benign disease course if undergoing early surgical intervention, with fewer admissions to hospital and a trend to reduced overall operation rates.

Abstract 4920: Evaluation of a diagnostic blood test for colorectal cancer screening and as a triage tool to stratify patients for colonoscopy

Worldwide, colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death, with an annual incidence in 2012 of 1.4 million CRC cases and an annual mortality around 700,000 [1]. Fortunately the majority of colorectal cancer (CRC) cases are preventable by early detection and removal by colonoscopy or surgery. Currently in Australia, the only widely used non-invasive screening test for CRC is the immunological faecal occult blood test (iFOBT), which has been shown to reduce CRC incidence and mortality by 15-25% [2]. There are however significant issues that limit its effectiveness as a diagnostic screening tool, such as a poor positive predictive value of 5.3% for suspected cancer and low participation rate of approximately 33.5% in 2013, as seen in the Australian National Bowel Cancer Screening Program (NBCSP)[2]. Evidence indicates a robust, blood-based, diagnostic assay would increase screening participation and compliance. In our systematic analysis of 68 individual biomarkers using logistic regression strategies, we have identified a unique 3 protein biomarker panel blood test consisting of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2)[3]. This panel not only differentiates between CRC and normal patient sera with 95% specificity and 73% sensitivity in a single measurement (c.f. FOBT: 35-50 sensitivity [4]) but early stage disease. Currently, we are prospectively collecting a new cohort of over 1,000 blood samples from volunteers that have undergone colonoscopy, that include CRC patients, controls and other pathologies (IBD, other cancers) to specifically determine the performance of this blood test in asymptomatic and surveillance screening populations. We will also evaluate directly the accuracy and performance of our biomarker panel, as a diagnostic test suitable for CRC screening versus the iFOBT used in the NBCSP, as an intermediary test in the triage of individuals with positive iFOBT result for need and urgency to have a colonoscopy. 1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.1, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 2. National Bowel Cancer Screening Program: monitoring report 2012-2013. AIHW & Australian Government Department of Health and Ageing, 2014. 3. Fung KYC et al 2015. Blood-based protein biomarker panel for the detection of colorectal cancer. PloS one. 2015;10(3):e0120425. 4. Morikawa, T et al., A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology, 2005. 129(2): p. 422-8. Citation Format: Leah J. Cosgrove, Kathy Surinya, Kim YC Fung, Ilka Priebe, Mike Buckley, Tim Adams, Bruce Tabor, Leanne Purins, Trevor Lockett, Hugo Leroux, Peter Gibbs, Jiangqin Wei, Ed Nice, Tony Burgess, Michelle Thomas, James Moore, Raj Singh, Andrew Ruszkiewicz. Evaluation of a diagnostic blood test for colorectal cancer screening and as a triage tool to stratify patients for colonoscopy. [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 4920.