WMV - 9857K, Video of scr-siRNA treated A549 cell growth.

(8129 KB) - Bar chart of scoring of immunohistochemistry and representative immunohistochemistry images of HT29 xenografts with and without acetazolomide and/or Bevacizumab treatment. CAIX expression (vehicle (DMSO) *p<0.05, n=5; ATZ **p<0.01, n=5) and CD31 (vehicle (DMSO) *p<0.05, n=5; ATZ **p<0.01, n=5) were significantly different between PBS and Bevacizumab treated xenografts. The amount of necrosis was significantly different between vehicle and ATZ treated xenografts (***P<0.001, n=5). CAIX, and CD31 expression staining is dark brown. H&E, dark pink denotes viable tissue, light pink denotes necrosis. N denotes areas of necrosis. Arrows point to positive staining. Scale bars represent 200m on 5x images and 100m on 20x images.

(1543 KB) - Analysis of the viable tumour volume at the first endpoint of the HCT116 and HT29 xenograft and spheroid experiments revealed that the CAIX expressors had increased viable tissue in HCT116 (A) xenografts (40% more viable tissue at day 14, ***p<0.01, n=10) and (B) spheroids (16% more viable tissue at day 13, **p<0.01, n=5) and HT29 (C) xenografts (34% more viable tissue at day 20, p<0.05, n=5) and (D) spheroids (45% more viable tissue at day 16, *p<0.01, n=5).

Supplementary Methods, Figure Legends 1-6, Tables 1-6 from The Histone Demethylase JMJD2B Is Regulated by Estrogen Receptor α and Hypoxia, and Is a Key Mediator of Estrogen Induced Growth

(244 KB) - Bar chart of scoring of immunohistochemistry and representative immunohistochemistry images of HT29 shCA9 and EV xenografts with and without Bevacizumab treatment. CAIX expression (**p<0.01 ***p<0.001, n=5), proportion of necrosis (*p<0.05, n=5) and cleaved caspase 3 expression (*p<0.05, n=5) were significantly different between EV and shCA9. CAIX expression (EV ***p<0.001, n=5), HIF1 (shCA9 *p<0.05, n=5, EV**p<0.01, n=5), cleaved caspase 3 expression (shCA9 **p<0.01, n=5, EV *p<0.05, n=5) and CD34 (shCA9 *p<0.05, n=5, EV *p<0.05, n=5) expression were significantly different between Bevacizumab treated and untreated xenografts. CAIX, HIF1, cleaved caspase 3, KI67 and CD34 expression staining is dark brown. H&E, dark pink denotes viable tissue, light pink denotes necrosis. N denotes areas of necrosis. Arrows point to positive staining. Scale bars represent 200m on 5x images and 100m on 20x images.

(1056 KB) - Analysis of CA9 RNA expression in U87 xenografts untreated and with chronic Bevacizumab treatment. Bevacizumab treatement increased CA9 expression (***p<0.001, n=5).

Supplementary Figures 1-6 from The Histone Demethylase JMJD2B Is Regulated by Estrogen Receptor α and Hypoxia, and Is a Key Mediator of Estrogen Induced Growth

This chapter contains sections titled: Clinical features Histopathology, immunophenotype and molecular genetics Treatment and Prognosis

Immunohistology is the microscopic study of cells and tissues using specific antibodies that bind to individual molecules expressed by the cellular and non-cellular components of the tissues. This branch of science is an essential link in the analysis and interpretation of data from high throughput genomic and proteomic technologies. Its use, both in the research and in the clinical arenas, has led to an increased understanding of cancer biology. This knowledge has also resulted in improvements in diagnosis, the provision of prognostic and predictive information, and highlighted the use of appropriate treatments. Furthermore, immunohistochemistry is a critical component in the search for personalized treatments. The ongoing advances in the availability of specific validated antibodies, continued improvements in staining and image analysis, and the integration of different technologies will ensure that immunohistochemistry becomes an even more essential tool in the study of cancer biology.

Solid tumours need a blood supply, and a large body of evidence has previously suggested that they can grow only if they induce the development of new blood vessels, a process known as tumour angiogenesis. On the basis of this hypothesis, it was proposed that anti-angiogenic drugs should be able to suppress the growth of all solid tumours. However, clinical experience with anti-angiogenic agents has shown that this is not always the case. Reports of tumours growing without the formation of new vessels can be found in the literature dating back to the 1800s, yet no formal recognition, description and demonstration of their special biological status was made until recently. In 1996, we formally recognized and described non-angiogenic tumours in lungs where the only blood vessels present were those originating from normal lung tissue. This is far from an isolated scenario, as non-angiogenic tumour growth has now been observed in tumours of many different organs in both humans and preclinical animal models. In this Opinion article, we summarize how these tumours were discovered and discuss what we know so far about their biology and the potential implications of this knowledge for cancer treatment.

Background ECG strain pattern is frequently presented in AS patients as a marker of LV hypertrophy and is emerging as one of the predictors for AVR. Our previous study had demonstrated that the normalisation of ST depression occurred within first 24 hours after AVR for AS while that of inverted T wave took 6–12 months in parallel to the time course of LVH regression. We hereby studied myocardium gene expression profiling and tested the hypothesis that different molecular mechanisms could have underlined the ST depression and T wave inversion. Material and methods We studied 17 AS patients with age 73±8.5 years and 12 males. LV biopsy was taken during AVR. Myocardial gene expression profiling was studied using Stanford Human Exonic Evidence Based Oligonucleotide (HEEBO) array. T wave inversion was defined by mean voltage of T wave in ECG lead I, aVL, V5 and V6 below −50uV (T-invert, n=8; T-norm, n=9). ST depression was defined by a mean value of ST in the same leads being below –50 uV (ST-dep, n=9; ST-norm, n=8). Differential gene expression profiling within ST and T wave sub-group were carried out by parametric permutative (permutation times=1000) t-test using the p-value cut-off 0.01 respectively. Further gene functional annotation clusters were performed using Database for Annotation, Visualisation and Integrated Discovery (DAVID, NIH) and pathway analysis of GeneSpring GX10. Results We identified 588 differentially expressed genes (DEGs) between the T-invert and T-norm with 308 genes up-regulated and 280 genes down-regulated in T-invert. Using Direct Interaction Network, genes with more intensive interactions were PLCL1, FGF1, MGP, FABP4, IL31 and IFI27 in up regulation and EGF, PAK2 SYT1, PPA1 and CSH1 in down-regulation, in T-invert. Between ST-dep and ST-normal, we identified 202 DEGs, with 95 up and 107 down regulated in ST-dep. Using Direct Interaction Network analysis, we identified interactive connexion of genes, including ARHGEF1, ARRB1, PPM1A, CD28, SNX9, PRSS27, ASXL1, HDAC9 and BCOR, with no centre nodes orientation. The Significantly Enriched Core Pathways Analysis and DAVID gene functional annotation clustering further revealed that the top pathways underlined the genes of T-invert were oxidative phosphorylation, mitochondria, cell cycle/mitosis, cell adhesion, EGF/ERBB pathway, fatty acid metabolism; whilst for the genes of ST-dep, the main pathways were G protein signalling pathway, transcription regulation of adipocyte differentiation and membrane trafficking/endocytosis. Conclusions Our study provides new molecular insights into ECG strain pattern in LV hypertrophy due to AS. T wave inversion appears to be involved in down regulation of energy metabolism and cell cycle but ST-depression is mainly associated with changes in signalling transduction. Understanding these different biological significance will help a more precise clinical interpretation of ECG strain pattern and its changes following AVR for AS.

Whole-genome sequencing (WGS) has transformed the understanding of the genetic drivers of cancer and is increasingly being used in cancer medicine to identify personalized therapies. Here we describe a case in which the application of WGS identified a tumoral BRCA2 deletion in a patient with aggressive dedifferentiated prostate cancer that was repeat-biopsied after disease progression. This would not have been detected by standard BRCA testing, and it led to additional treatment with a maintenance poly ADP ribose polymerase (PARP) inhibitor following platinum-based chemotherapy. This case demonstrates that repeat biopsy upon disease progression and application of WGS to tumor samples has meaningful clinical utility and the potential to transform outcomes in patients with cancer.

Background Left ventricular hypertrophy (LVH) assessment by ECG voltage or image based mass volume is commonly used in clinical practice and research. Our previous study of LVH regression after AVR for AS demonstrated a more complete regression in ECG voltage than in LV mass, thus indicated that ECG and Echo could be quantifying different aspects of LVH. In this study, we used gene expression profiling to examine whether different molecular pathways are involved in LV hypertrophy defined by ECG voltage or Echo LV mass. Material and Methods We studied 17 patients with aortic stenosis, aged 73±8.5 years with 12 males. Gene expression profiling of LV myocardium biopsy during AVR was carried out using Stanford Human Exonic Evidence Based Oligonucleotide (HEEBO) array. Using ECG QRS voltage cut-off 3.0 mv, 8 patients had LVH (ECG-LVH) and 9 had not (ECG-Norm ). Using Echo LV mass index 125 g/m2 for man and 105 g/m2 for woman, 9 cases had LVH (Echo-LVH) and 8 had not (Echo-Norm). The gene expression profiling comparisons were carried out between ECG-LVH vs. ECG-Norm as well as Echo-LVH vs. Echo-Norm, respectively, by parametric permutative (permutation times=1000) t-test using the p-value cut-off 0.01. Further gene functional annotation clusters were performed using Database for Annotation, Visualisation and Integrated Discovery (DAVID, NIH) and pathway analysed by GeneSpring GX10. Results There are 163 genes significantly differentially expressed (DEGs) between ECG-LVH and ECG-Norm. Among which, 81 genes were up-regulated and 82 genes down-regulated in ECG-LVH. Between Echo-LVH and Echo-Norm, 256 DEGs were identified, of which 141 genes were up and 115 genes were down regulated in Echo-LVH. The Direct Interaction Network of 163 DEGs in ECG-LVH revealed several connected island hubs with genes of HGF, CDK5RAP2, FYN (up-regulated) and IL6, PTBP2 and IRF1 (down-regulated). Of 257 DEGs in Echo-LVH, there were more abundant island-like connexions. The hubs of the connexion include PTEN, GNL3, AREG, ADAM17 and PPP1CA (up-regulated) and DCTN6, TWIST1 and ITGAM (down regulated). Further data mining and analysis of the two DEGs lists using DAVID and the Significantly Enriched Core Pathways analysis has demonstrated that the DEGs of ECG-LVH and ECHO-LVH have similar regulation directions in pathways/annotation clustering, including transcription regulation, nucleotide binding, zinc finger, focal adhesion. In addition, ECG-LVH has down regulation in wounding healing/inflammatory/cytokines pathway indicating a lower interstitial fibrosis activity compared with ECG-Norm; In Echo-LVH, mitochondrial protein importing, metalloprotease/ECM remodelling and cell division pathways were up-regulated compared to Echo-Norm. Conclusions Cardiac hypertrophy diagnosed by ECG voltage or Echo muscle mass share many common molecular pathways that have been implicated in LVH. The unique pathways associated with ECG voltage based LVH imply a predominant hypertrophy of myocytes; in contrast, Echo mass based LVH involves the unique pathways of myocardial energy and ECM remodelling. The convergence and divergence of underlying molecular pathways between ECG based and Echo based LVH suggest the two methods may provide complimentary clinical value in the assessment of LVH and its response to treatment.

Purpose: To identify hypoxia-related biomarkers indicative of response and resistance to epirubicin treatment in patients with locally advanced breast cancer. Patients and Methods: One hundred seventy-six women with T2-4 N0-1 breast tumours were randomly assigned to receive epirubicin 120 mg/m2/1-21 (EPI ARM), epirubicin 120 mg/m2/1-21 + erythropoietin 10.000 IU sc three times weekly (EPIEPO ARM) and epirubicin 40 mg/m2/w-q21 (EPI-W ARM). Sixteen tumour proteins involved in cell survival, hypoxia, angiogenesis and growth factor, were assessed by immunohistochemistry in pre-treatment samples. A multivariate generalized linear regression approach was applied using a penalized least-square minimization to perform variable selection and regularization. Results: VEGF and GLUT-1 expression were significantly positively associated with complete response (CR) to treatment in all leave-one-out iterations. Bcl-2 expression was inversely correlated with pCR, whilst EPO expression was positively correlated with pathological complete response (pCR). Haemaglobin and HIF-1a nuclear expression were inversely correlated with pCR. HB and HIF-1a expression were associated with a higher risk of relapse and overall survival. Conclusion: Hypoxic biomarkers determines the epirubicin resistance in breast cancer. Assessment of such biomarkers, may be useful for predicting chemosensitivity and also anthracycline-based treatment outcome.

Patients with encapsulated colorectal liver metastases (CRLM) have a better prognosis than those without a capsule. The reason for the encapsulation is unknown. Hypoxia inducible factor-1α (HIF-1α) increases tumor angiogenesis and tumor tissue expression is associated with reduced survival. Our aim was to determine whether the good prognosis of encapsulated CRLM is associated with reduced HIF-1α expression by the cancer.The study selected only patients who had not undergone neoadjuvant chemotherapy prior to a potentially curative hepatectomy for CRLM. From 30 selected patients, serial sections were cut from a single randomly selected metastasis. Morphology was assessed following H&E staining. Tumor hypoxia, vascular endothelial growth factor (VEGF), proliferation, and microvascular density (MVD) were assessed by immunostaining for HIF-1α and carbonic anhydrase-9 (CA-9), VEGF, Ki67, and cluster of differentiation-31, respectively. MVD was calculated in the vascular hot spots. Pathology was reported without clinical outcome information. Actual long-term survival was recorded.Thirteen (43%) of the cancers were encapsulated CRLM containing glands which were large, complex, and cribriform. Thirteen (43%) were infiltrative CRLM and their glands were small, closely packed, and rounded with vessels in the interglandular fibrous tissue with no capsule; 3 (10%) had a mixed picture. Encapsulated CRLM had a higher expression of HIF-1α (58% vs 8%, P = 0.03), CA-9 (42% vs 0%, P = 0.04), and VEGF (92% vs 25%, P = 0.02). MVD was lower in the encapsulated CRLM group (37 mm vs 143 mm, P < 0.001). The median follow-up was 115 months. The encapsulated CRLM group had a better overall and 5-year survival (relative hazard: 0.58, P = 0.057 and hazard ratio: 0.52, P = 0.044).There are 2 main morphological appearances of CRLM which have very different long-term survival following liver resection surgery. The morphology is associated with differences in expression of HIF-1α, CA-9, VEGF, and angiogenesis.

BACKGROUND:Angiogenesis is not essential for tumours to develop and expand, as cancer can also grow in a non-angiogenic fashion, but why this type of growth occurs is unknown. Surprisingly, our data from mRNA transcription profiling did not show any differences in the classical angiogenic pathways, but differences were observed in mitochondrial metabolic pathways, suggesting a key role for metabolic reprogramming. We then validated these results with mRNA profiling by investigating differential protein expression via immunohistochemistry in angiogenic and non-angiogenic non-small cell lung cancers (NSCLCs). METHODS:Immunohistochemical staining for 35 angiogenesis- and hypoxia-related biomarkers were performed on a collection of 194 angiogenic and 73 non-angiogenic NSCLCs arranged on tissue microarrays. Sequencing of P53 was performed with frozen tissue samples of NSCLC. RESULTS:The non-angiogenic tumours were distinguished from the angiogenic ones by having higher levels of proteins associated with ephrin pathways, mitochondria, cell biogenesis, and hypoxia-inducible factor 1 (HIF1) regulation by oxygen and transcription of HIF-controlled genes but lower levels of proteins involved in the stroma, cell-cell signaling and adhesion, integrins, and Delta-Notch and epidermal growth factor (EGF)-related signaling. However, proteins classically associated with angiogenesis were present in both types of tumours at very comparable levels. Cytoplasmic expression of P53 was strongly associated with non-angiogenic tumours. A pilot investigation showed that P53 mutations were observed in 32.0% of angiogenic cases but in 71.4% of non-angiogenic tumours. CONCLUSIONS:Our observations thus far indicate that both angiogenic and non-angiogenic tumours experience hypoxia/HIF and vascular endothelial growth factor (VEGF) pathway protein expression in a comparable fashion. However, angiogenesis does not ensue in the non-angiogenic tumours. Surprisingly, metabolic reprogramming seems to distinguish these two types of neoplastic growth. On the basis of these results, we raise the hypothesis that in some, but not in all cases, initial tissue remodeling and/or inflammation could be one of the secondary steps necessary to trigger angiogenesis. In the non-angiogenic tumours, in which neovascularisation fails to occur, HIF pathway activation could be the driving force toward metabolic reprogramming.

Although sorafenib is approved for the treatment of hepatocel-lular carcinoma (HCC), the ability of this anti-angiogenic tyro-sine kinase inhibitor to extend survival in HCC patients is limited because of acquired drug resistance, whose mechanism is poorly understood. In this issue of the Journal, Kuczynski and colleagues (1) report a mechanism of acquired resistance to sorafenib using an orthotopic HCC xenograft model. The authors tell us how, although these HCC are initially angiogenic and re-spond to sorafenib, resistance occurs because the neoplastic cells switch to a non-angiogenic phenotype and actively co-opt the normal liver vasculature instead of inducing angiogenesis. These results have important implications for understanding this particular form of resistance to anti-angiogenic drugs and the biology of the tumors that can develop such a resistance. The non-angiogenic growth of tumors has already been hy-pothesized to be one of the possible reasons for intrinsic and/or acquired cancer resistance to anti-angiogenic treatment (2). In this paper, it is now proven that an angiogenic malignancy can, following treatment with a drug inhibiting sprouting angiogenesis, switch to a non-angiogenic phenotype in order to keep growing. This finding further supports notion that neoplasia is some tumors non-angiogenic many more can a

The limited clinical benefits from current antiangiogenic therapy for cancer patients have triggered some critical thoughts and insightful investigations aiming to further elucidate the relationship between vessels and cancer. Tumors need blood perfusion but there are mounting evidences that angiogenesis alone does not explain it in all the neoplasms. In this editorial, for a special issue on tumor and vessels published in the Chinese Journal of Cancer, we briefly introduce the history of the evidences that solid tumors can sometimes obtain blood perfusion by alternative approaches other than sprouting angiogenesis, i.e., vessel co-option and vasculogenic mimicry. This editorial provides also the links to several most recently published discoveries and hypotheses on tumor interaction with blood vessels.

Introduction: PVE facilitates hepatectomy in patients with a small anticipated future liver remnant, but stimulates tumour growth. Hypoxia may mediate this increased tumour growth as Hypoxia-Inducible-Factor-1α (HIF-1α) increases angiogenesis, invasion and other Hallmarks of Cancer. This study investigates the relationship of patient survival to expression of HIF-1α and other hypoxia factors regulated in resected CRLM, in a case control study, comparing patients with and without PVE. Methods: Twenty-six patients who had PVE were compared with 25 controls matched with the number and size of metastases (tumour-burden). Immunostaining was performed on CRLM formalin-fixed-paraffin-embedded sections to compare the expression of hypoxic factors regulated factors, HIF-1α and CA-9, vascular endothelial growth factor (VEGF) and a blood vessel marker, CD31, between the groups. Disease progression, liver specific recurrence and actuarial survival were recorded. Results: The clinicopathological characteristics of the cancers were comparable between the groups. The median follow-up was 115 months (range 106–124). Overall, 5 year, local hepatic recurrence-free survival and progression-free periods were poor in those patients undergoing PVE (p = 0.026, p = 0.060, p = 0.001 and p = 0.008 respectively). The expression of hypoxia markers between the groups was similar. Conclusion: This is the first long-term (>5 year) case matched series on outcome of patients with CRLM following PVE. Whilst, PVE facilitates potentially curative resection of CRLM, prognosis is less than those patients not requiring PVE. Whilst further molecular research is required to investigate the cause of tumour growth and reduced prognosis, the hepatic artery buffer response may have a role in preventing intratumoural hypoxia following PVE.