Sirwan M. Hadad

Targeting AMPK: A new therapeutic opportunity in breast cancer

BACKGROUND This paper reviews the mammalian Target Of Rapamycin (mTOR) pathway dysregulation in breast cancer, and the current evidence targeting this pathway directly or through activation of AMP-activated protein kinase (AMPK) as an additional therapeutic opportunity for intervention in breast cancer. METHODS Relevant articles were identified through computerised searches of Medline and Pubmed. Secondary articles were identified from the reference lists of key papers and by hand searching. RESULTS AND CONCLUSION The current consensus to target the AMPK/mTOR pathway in breast cancer is based on in vitro and epidemiological evidences. A low incidence of cancer in diabetic patients on metformin has been explained in vitro by the drugs anti-proliferative effect through activation of AMPK. There is a need to explore the anticancer effects of metformin and the potential to develop the therapeutic avenues offered by targeting the AMPK/mTOR pathway.

Histological evaluation of AMPK signalling in primary breast cancer

BackgroundAMP-activated protein kinase (AMPK) acts as a cellular fuel gauge that responds to energy stress by suppressing cell growth and biosynthetic processes, thus ensuring that energy-consuming processes proceed only if there are sufficient metabolic resources. Malfunction of the AMPK pathway may allow cancer cells to undergo uncontrolled proliferation irrespective of their molecular energy levels. The aim of this study was to examine the state of AMPK phosphorylation histologically in primary breast cancer in relation to clinical and pathological parameters.MethodsImmunohistochemistry was performed using antibodies to phospho-AMPK (pAMPK), phospho-Acetyl Co-A Carboxylase (pACC) an established target for AMPK, HER2, ERα, and Ki67 on Tissue Micro-Array (TMA) slides of two cohorts of 117 and 237 primary breast cancers. The quick score method was used for scoring and patterns of protein expression were compared with clinical and pathological data, including a minimum 5 years follow up.ResultsReduced signal, compared with the strong expression in normal breast epithelium, using a pAMPK antibody was demonstrated in 101/113 (89.4%) and 217/236 (91.9%) of two cohorts of patients. pACC was significantly associated with pAMPK expression (p = 0.007 & p = 0.014 respectively). For both cohorts, reduced pAMPK signal was significantly associated with higher histological grade (p = 0.010 & p = 0.021 respectively) and axillary node metastasis (p = 0.061 & p = 0.039 respectively). No significant association was found between pAMPK and any of HER2, ERα, or Ki67 expression, disease-free survival or overall survival.ConclusionThis study extends in vitro evidence through immunohistochemistry to confirm that AMPK is dysfunctional in primary breast cancer. Reduced signalling via the AMPK pathway, and the inverse relationship with histological grade and axillary node metastasis, suggests that AMPK re-activation could have therapeutic potential in breast cancer.

A candidate molecular signature associated with tamoxifen failure in primary breast cancer

IntroductionFew markers are available that can predict response to tamoxifen treatment in estrogen receptor (ER)-positive breast cancers. Identification of such markers would be clinically useful. We attempted to identify molecular markers associated with tamoxifen failure in breast cancer.MethodsEighteen initially ER-positive patients treated with tamoxifen requiring salvage surgery (tamoxifen failure [TF] patients) were compared with 17 patients who were disease free 5 years after surgery plus tamoxifen adjuvant therapy (control patients). cDNA microarray, real-time quantitative PCR, and immunohistochemistry on tissue microarrays were used to generate and confirm a gene signature associated with tamoxifen failure. An independent series of 33 breast tumor samples from patients who relapsed (n = 14) or did not relapse (n = 19) under tamoxifen treatment from a different geographic location was subsequently used to explore the gene expression signature identified.ResultsUsing a screening set of 18 tumor samples (from eight control patients and 10 TF patients), a 47-gene signature discriminating between TF and control samples was identified using cDNA arrays. In addition to ESR1/ERα, the top-ranked genes selected by statistical cross-analyses were MET, FOS, SNCG, IGFBP4, and BCL2, which were subsequently validated in a larger set of tumor samples (from 17 control patients and 18 TF patients). Confirmation at the protein level by tissue microarray immunohistochemistry was observed for ER-α, γ-synuclein, and insulin-like growth factor binding protein 4 proteins in the 35 original samples. In an independent series of breast tumor samples (19 nonrelapsing and 14 relapsing), reduced expression of ESR1/ERα, IGFBP4, SNCG, BCL2, and FOS was observed in the relapsing group and was associated with a shorter overall survival. Low mRNA expression levels of ESR1/ERα, BCL2, and FOS were also associated with a shorter relapse-free survival (RFS). Using a Cox multivariate regression analysis, we identified BCL2 and FOS as independent prognostic markers associated with RFS. Finally, the BCL2/FOS signature was demonstrated to have more accurate prognostic value for RFS than ESR1/ERα alone (likelihood ratio test).ConclusionsWe identified molecular markers including a BCL2/FOS signature associated with tamoxifen failure; these markers may have clinical potential in the management of ER-positive breast cancer.

Delay from Symptom Onset Increases the Conversion Rate in Laparoscopic Cholecystectomy for Acute Cholecystitis

BackgroundRandomized trials suggest that laparoscopic cholecystectomy should be performed on first admission for acute cholecystitis. However, this is not widely practiced, possibly because of a perceived high conversion rate. We hypothesized that delay from onset of symptoms may increase the conversion rate.MethodsWe performed a retrospective case note review of patients undergoing emergency cholecystectomy in a single institution between January 2002 and December 2005. We analyzed whether delay from onset of symptoms was related to the conversion rate in patients with a histopathological diagnosis of acute cholecystitis.ResultsOf patients who underwent emergency laparoscopic cholecystectomy in our institution, 32.4% (197/608) had acute cholecystitis on histopathology. The conversion rate of those with acute cholecystitis was considerably higher (24.4%) than for those with other pathologies (6.3%). For patients with acute cholecystitis, the conversion rates increased with duration of symptoms: 9.5%, 16.1%, 38.9%, and 38.6% for delays of 0–2 days, 3–4 days, 5–6 days, and > 6 days from symptom onset, respectively (chi-square for trend = 14.27, DF = 1, p = 0.00016). Most conversions were due to the presence of acute inflammatory adhesions.ConclusionsEarly intervention for acute cholecystitis (preferably within 2 days of onset of symptoms) is most likely to result in successful laparoscopic cholecystectomy; increasing delay is associated with conversion to open surgery.

Phase II Randomized Preoperative Window-of-Opportunity Study of the PI3K Inhibitor Pictilisib Plus Anastrozole Compared With Anastrozole Alone in Patients With Estrogen Receptor–Positive Breast Cancer

PURPOSE Preclinical data support a key role for the PI3K pathway in estrogen receptor-positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. PATIENTS AND METHODS In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. RESULTS There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination:anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P = .03); for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. CONCLUSION Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer.

Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERα negative MDA-MB-435 breast cancer model

MDA-MB-435 cell line has been used for decades as a model of metastatic human breast cancer [1]. This cell line was derived at M.D. Anderson in 1976 from a pleural effusion from a 31-year old woman with a history of breast cancer [2, 3]. However, recent advances in gene expression analysis, which allow the opportunity to more fully characterize tumour cell lines, revealed that the pattern of gene expression for MDA-MB-435 is more closely resembles melanoma cell lines than breast tumour lines [4]. These findings prompted Ellison et al. to undertake a more detailed study of the characteristics of MDA-MB-435 [5]. In brief, they confirmed that breast-specific genes were not detectably expressed in MDA-MB-435 compared to most of the breast tumour cell lines they were investigating. Furthermore, melanocyte-specific genes were expressed in MDAMB-435, as well as in most of the other melanoma cell lines, but were not detectable in the other breast tumour cell lines. Additionally, xenografts of MDA-MB-435 implanted into mammary fat pads of female Severe Combined Immunodeficiency (SCID) mice showed immunohistochemical staining consistent with melanocytic origin. Furthermore, Rae et al. published an article in Breast Cancer Research and Treatment last year concluded ‘‘All currently available stocks of MDA-MB-435 cells are derived from the M14 melanoma cell line and can no longer be considered a model of breast cancer’’ [6]. They used karyotype, Comparative Genomic Hybridization (CGH), and microsatellite polymorphism analyses, combined with bioinformatics analysis of gene expression and SNP data to test the hypothesis that the MDA-MB-435 cell line is derived from the melanoma cell line M14.

The Effect of Neoadjuvant Metformin on Breast Cancer Related Genes Clinical Trial – A Preliminary Result.

Other authoring group: Dougal Adamson, Lee Baker, Douglas C Brown, Sally Chalmers, Amanda Degabriele, Janet Flinn, Kenneth Fowler, Avril Gunning, Grahame Hardie, Julie Lindsay, Gillian Little, Denis McLean, Robert Murdoch, Colin Purdie, Marta Reis, Valerie Walker. Introduction Metformin, used in the treatment of diabetes for 50 years, has been linked to a reduced incidence of breast cancer and to an enhanced pathological response when administered concurrent with neoadjuvant chemotherapy. It has been postulated that the anti-neoplastic effect of metformin is through activation of AMP-activated protein Kinase (AMPK). The aim of this open label, randomised clinical trial was to investigate the effect of neoadjuvant metformin on gene expression in primary breast cancer. Methods Non-diabetic women with histologically proven, operable, primary invasive breast cancer of ≥1cm in size were offered the trial following regional Research Ethics Committee approval.The trial had 2 arms, A and B: in arm A (8 patients), patients had core biopsies at three time points: one at presentation; one after a week of no treatment (internal control); and a further biopsy at the time of definitive surgery after at least 2 weeks of metformin. In trial arm B (47 patients), patients had core biopsies at two time points: one before randomisation to either take metformin or not, and one at the time of definitive breast surgery. Metformin was given as 500mg o.d. orally for one week, increased to 1g b.d. orally for at least another week and until the day prior to excisional breast surgery.RNA was extracted from formalin fixed cores and examined using a Disease-Specific Array for breast cancer on an Affymetrix platform (ALMAC Diagnostics Group, Craigavon, UK). Subsequent confirmatory polymerase chain reaction and immunohistochemistry techniques were used to examine the histological material. Results Type of results expected: [Change of target gene expression in response to neoadjuvant metformin; symptomatic drug toxicities; potential for therapeutic effects of metformin in non-diabetic patients in vivo.] Results from the first arm: The triplet of core biopsies from 6/8 (75%) of the A arm patients passed the GeneGhip QC and data integrity assessment. Patients were 41-65 (mean: 53) years, with IDC NST, grade II-III, and all ER positive. 270 genes were differentially expressed significantly by wounding, and 63 by metformin. Among the genes significantly over-expressed by metformin were AMPK-beta and PI3K; and those down regulated by metformin were AMPK-gamma and ADCY1. Samples from the B arm patients are in analysis. Conclusion The gene expression changes identified in response to neoadjuvant metformin suggest that metformin improves DNA damage recognition and repair and regulates cancer cell metabolism. These results suggest mechanisms by which metformin could be efficacious in the treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3145.

αB-crystallin, vimentin and increased p53 expression levels in breast cancer is associated with poor prognosis.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #5070 Background: Previous studies have identified αB-crystallin as a marker of poor prognosis for breast cancer and have suggested that it is an excellent marker for tumours of basal origin. Increased expression of αB-crystallin has been associated with anchorage-independent growth and metastasis leading to the suggestion that αB-crystallin is an oncoprotein. We have considered whether the αB-crystallin binding proteins, vimentin and HSP27 also show a similar association with poor prognosis. We also investigated whether either HSP27 phosphorylation HSP27 or p53 expression could be associated with αB-crystallin expression. Methods: Tissue Micro Arrays of 0.6x0.6mm cores of 205 breast cancers were stained with antibodies to αB-crystallin, vimentin, p53 (antibodies DO1, FP3), HSP27 (antibody ERD5) and HSP27 82P. The levels of protein expressions were then compared with clinical and pathological parameters. Results: The expression of αB-crystallin was positively associated with vimentin (P<0.001 Fishers exact test (FET)). We have confirmed that αB-crystallin expression is linked to a low expression of the estrogen receptor and reduced survival (P<0.001 (FET), P=0.002 Kaplan Meier Log Rank (KM) respectively). Vimentin expression was similarly associated with estrogen receptor (ER) negative cancers and poor survival (P <0.001 (FET), P= 0.002 (KM Log Rank) respectively). Low expression of HSP27 resulted in poor three-year survival (P=0.007 (KM Log Rank)), but longer-term survival was unaffected (P=0.09 (KM Log Rank)). In contrast to αB-crystallin, low expression of HSP27 was associated with ER and progesterone receptor (PGR) negativity (P<0.001 (FET), P=0.001 (FET)). HSP27 82P similarly resulted in poor three-year survival (P=0.01 (KM Log Rank)) compared with longer-term survival (P=0.05 (KM Log Rank)). In samples positive for either αB-crystallin or vimentin, a strong association with increased p53 expression (p53 antibodies: DO1(P<0.001 (FET)) and FP3(P<0.001 (FET))) was found. Conclusions: The increased expression of the protein chaperone, αB-crystallin was linked with reduced survival as well as its binding partner, vimentin. We found that within this patient subgroup, there was an increased level of p53. The potential functional significance of this interaction for metastasis will be discussed in the context of other predictive markers for breast cancer. A similar association was not found for HSP27 expression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5070.

Delay from symptom onset increases the conversion rate in laparoscopic cholecystectomy for acute cholecystitis (World Journal of Surgery 31, 6, DOI: 10.1007/s00268-007-9050-2)

BACKGROUND Randomized trials suggest that laparoscopic cholecystectomy should be performed on first admission for acute cholecystitis. However, this is not widely practiced, possibly because of a perceived high conversion rate. We hypothesized that delay from onset of symptoms may increase the conversion rate. METHODS We performed a retrospective case note review of patients undergoing emergency cholecystectomy in a single institution between January 2002 and December 2005. We analyzed whether delay from onset of symptoms was related to the conversion rate in patients with a histopathological diagnosis of acute cholecystitis. RESULTS Of patients who underwent emergency laparoscopic cholecystectomy in our institution, 32.4% (197/608) had acute cholecystitis on histopathology. The conversion rate of those with acute cholecystitis was considerably higher (24.4%) than for those with other pathologies (6.3%). For patients with acute cholecystitis, the conversion rates increased with duration of symptoms: 9.5%, 16.1%, 38.9%, and 38.6% for delays of 0-2 days, 3-4 days, 5-6 days, and > 6 days from symptom onset, respectively (chi-square for trend = 14.27, DF = 1, p = 0.00016). Most conversions were due to the presence of acute inflammatory adhesions. CONCLUSIONS Early intervention for acute cholecystitis (preferably within 2 days of onset of symptoms) is most likely to result in successful laparoscopic cholecystectomy; increasing delay is associated with conversion to open surgery.