Accepted Manuscript
BCL9 Upregulation in Adrenocortical Carcinoma: A Novel Wnt/B-Catenin Activating Event Driving Adrenocortical Malignancy
Taylor C. Brown, MD, MHS, Norman G. Nicolson, MD, Reju Korah, PhD, Tobias Carling, MD, PHD, FACS
Journal of the American College of Surgeons
| PII: | S1072-7515(18)30105-4 |
| DOI: | 10.1016/j.jamcollsurg.2018.01.051 |
| Reference: | ACS 9057 |
| To appear in: | Journal of the American College of Surgeons |
| Received Date: | 3 October 2017 |
| Revised Date: | 21 December 2017 |
| Accepted Date: | 11 January 2018 |
Please cite this article as: Brown TC, Nicolson NG, Korah R, Carling T, BCL9 Upregulation in Adrenocortical Carcinoma: A Novel Wnt/B-Catenin Activating Event Driving Adrenocortical Malignancy, Journal of the American College of Surgeons (2018), doi: 10.1016/j.jamcollsurg.2018.01.051.
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BCL9 Upregulation in Adrenocortical Carcinoma: A Novel Wnt/B-Catenin Activating Event Driving Adrenocortical Malignancy
Taylor C Brown MD, MHS, Norman G Nicolson MD, Reju Korah PhD, Tobias Carling MD, PhD, FACS
Department of Surgery, Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, New Haven, CT
Disclosure Information: Nothing to disclose. Support for this study: This study was supported by a grant from the Ohse Research Grant.
Corresponding Author Address: Tobias Carling, MD, PHD, FACS Department of Surgery, Yale Endocrine Neoplasia Laboratory Yale University School of Medicine 333 Cedar Street, TMP FMB130A, PO Box 208062, New Haven, CT, 06520 Tel: 203-737-2036 Fax: 203-737-4067 Email: tobias.carling@yale.edu
Presented at the 98th Annual Meeting of the New England Surgical Society, Bretton Woods, NH, September 2017.
Running Head: BCL9 and Adrenocortical Carcinoma
Keywords: Adrenocortical Carcinoma, BCL9, Wnt/ß-Catenin signaling.
ABSTRACT
Background: B-Cell CLL/Lymphoma 9 (BCL9) is a recently described oncogene that promotes tumorigenesis via activation of the Wnt/B-Catenin signaling cascade. Though constitutively active Wnt/ß-Catenin signaling is a molecular hallmark of adrenocortical carcinoma (ACC), a potential role for BCL9 to promote Wnt/B-Catenin pathway dysregulation in adrenocortical tumorigenesis remains to be elucidated.
Study Design: This study involved a retrospective analysis at a tertiary academic referral center of 27 patients with adrenocortical tumors, including in vitro investigation of BCL9. Wnt signaling pathway PCR array analysis queried comparative mRNA expression profiles of canonical Wnt pathway components including BCL9. Real-time quantitative PCR determined BCL9 mRNA expression levels in tumor samples. BCL9 mRNA expression levels were evaluated for correlation with tumor characteristics. RNA interference (RNAi) gene silencing was performed in ACC cell lines SW-13 and NCI-H295R to test the role of BCL9 on clonal cell growth.
Results: BCL9 gene expression levels were found to be significantly elevated in ACC compared to normal adrenal tissue (p<0.05). Furthermore, a significant correlation was observed between BCL9 mRNA levels and the malignant status of adrenocortical tumors (p<0.05). RNAi gene silencing of BCL9 inhibited clonal cell growth of SW-13 cells (p<0.05), but not NCI-H295R cells, which carry a constitutively active ß-Catenin mutation.
Conclusions: BCL9 is overexpressed in malignant adrenocortical tumors and promotes clonal ACC cell growth. These findings suggest that BCL9 overexpression may serve as an alternative driver of constitutive Wnt/B-Catenin activation in ACC and could represent a potential molecular and diagnostic marker of tumor malignancy.
ABBREVIATIONS
| ACA | Adrenocortical adenoma |
| ACC | Adrenocortical carcinoma |
| BCL9 | B-Cell CLL/Lymphoma 9 |
| DMEM | Dulbecco's modified Eagle's medium |
| RNAi | RNA interference |
ACCEPTED MANUS CRIPT
INTRODUCTION
Adrenocortical carcinoma (ACC) is a rare cancer that generally portends a poor prognosis. ACC incidence has been estimated to occur at rates ranging between 1 to 2 cases per million persons per year, though its true rate of incidence is unknown. Tumors occur most frequently during the 4th through 6th decades of life (1), with ACC occurrence also observed in segments of the pediatric population, typically in association with germline TP53 mutations (2). Other hereditary syndromes have also been associated with ACC, including Beckwith- Wiedemann syndrome, multiple endocrine neoplasia type 1, and Lynch syndrome, as well as other tumor related syndromes, though data are limited and further analysis is warranted (1).
Patients usually present with symptoms resulting from autonomous and excessive hormone production and/or local tumor mass effect (3). A significant proportion of patients, however, are diagnosed incidentally while obtaining cross-sectional imaging for other reasons (1). Based on an analysis of the German ACC Registry, average tumor size was reported to be 11.6 cm and 33% of patients were diagnosed with metastatic disease at the time of presentation, with lung and liver being the most frequent sites of metastases (4). Tumor staging is based on the European Network for the Study of Adrenal Tumor (ENSAT) staging system in conjunction with histo-pathological determination of malignancy. Stage I and stage II tumors are confined to the adrenal bed, while stage III and stage IV tumors infiltrate the surrounding tissue, invade adjacent organs, invade the renal vein or the inferior vena cava, have lymph node metastases, or distant metastatic lesions (5).
The primary treatment modality for non-metastatic ACC is surgical resection with the intent of an R0 resection. Current adjuvant and salvage treatments regimens include Mitotane with or without various combinations of chemotherapy regimens. However, it was not until
recently that such regimens were tested in a randomized control trial (6). With recent progress made in identifying molecular aberrations associated with cancer formation, several studies have assessed various targeted modalities. Unfortunately, most of these treatments demonstrated minimal effect on tumor progression and survival (1). Though recent discoveries have helped elucidate our understanding of the genetic drivers of ACC, these discoveries have not yet translated into improved outcomes, and overall prognosis remains poor (7, 8).
The Weiss scoring system remains the primary pathological parameter utilized to make a tissue diagnosis when malignancy is not clearly evident and relies on an evaluation of a standard battery of histological findings that include mitotic rate, cell necrosis, and vascular invasion. The Weiss scoring system has its limitations and significant uncertainty can remain for tumors with indeterminate scores. Thus, the need to identify reliable molecular markers is paramount to provide more accurate tissue diagnosis (9). Furthermore, after an apparent RO resection of ACC, local recurrence rates can be high (10). Characterization of high-risk molecular features could help determine which patients are at increased risk of recurrence after surgery and who may benefit from adjuvant therapy and closer surveillance.
One of the well-described driving events in ACC tumorigenesis is dysregulation of the canonical Wnt/B-Catenin signaling pathway, frequently via mutations of the proto-oncogene CTNNB1 (B-catenin). Activation of ß-Catenin localizes the protein to the nucleus and promotes target gene transcription, which in turn induces tumor formation (11). BCL9 is a proto-oncogene that was initially discovered overexpressed in B-cell lymphoma as a result of recurrent gene translocations (12). BCL9 was subsequently found to be a positive regulator of the Wnt/B- Catenin signaling pathway, serving as a critical adaptor protein for gene transcription (13). More recently, overexpression of BCL9 has been observed to be associated with tumor formation in
multiple cancers types, including hepatocellular, renal cell, breast, and colorectal cancers (14- 17). The potential role of BCL9 and its contribution to the activation of Wnt/B-catenin signaling in ACC has not been previously studied.
METHODS
Study cohort. Following approval by the Yale University institutional review board, 10 cases of histologically confirmed ACCs and 17 cases of adrenocortical adenomas (ACA) were selected for biochemical and clinical analysis. Post resection, the samples were isolated, dissected, histopathology confirmed via microscopic observation by an experienced endocrine pathologist, and collected fresh-frozen for further laboratory assays. Clinical characteristics of the patients are shown in Table 1. All fresh frozen adrenal tissues samples were maintained in a prospectively maintained endocrine tumor repository and an experienced endocrine pathologist reviewed tissues sections for confirmation of the diagnosis prior to investigation.
Transcription factor expression array. Transcription factor gene expression analysis was performed using the RT2 Profile PCR Array Human WNT Signaling Pathway (Qiagen) per the manufacturer’s instructions. Briefly, 100 ng of DNA-free RNA from 6 ACC tumor samples was purified using the RNeasy plus mini kit, subjected to cDNA synthesis using the RT2 first strand kit, amplified using RT2 SYBR Green Mastermix (All reagents from Qiagen), and subjected to real-time PCR analysis using a CFX96 Real-Time System thermo cycler (Bio-Rad). Relative expression levels were analyzed with SABioscience PCR Array Data Analysis web- based software on data web portal at www.SABiosciences.com/pcrarraydataanalysis.php. Three normal samples (histologically normal and adjacent to the tumor) were used for the array comparison.
Gene expression analysis. RNA was isolated from fresh frozen samples using the AllPrep DNA/RNA/Protein Kit (Qiagen). Quantity and quality of isolated RNA was assessed by spectrophotometry (NanoDrop Technologies, Inc) and two hundred ng of RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Bio-Rad). Real-time quantitative PCR was performed on a CFX96 Real-Time System thermo cycler (Bio-Rad) using TaqMan PCR master mix with primers and probes (Applied Biosystems) specific to BCL9 (Hs00979216_m1) and housekeeping gene large ribosomal protein 0 (RPLP0; Hs00420895 gH). Multiple tissues samples were assayed to account for tumor heterogeneity. Relative expression levels were calculated using the Livak method (18).
Immunohistochemistry. Four 5 um-thick representative sections of histologically confirmed ACAs, ACCs, and normal adrenal tissue from formalin fixed paraffin embedded tissue samples were selected for study. Using standard immunohistochemistry protocols (19), target epitopes were detected using rabbit anti-BCL9 polyclonal antibody (Santa Cruz Biotechnology) followed by goat anti-rabbit HRP conjugated monoclonal secondary antibody (Invitrogen). 3,3’-diaminobenzidine tretrachydrochloride (DAB) was utilized for antigen detection (Life Technologies). Sections were counterstained with hematoxylin and mounted using immunohistomount (Santa Cruz Biotechnology).
Cell culture and RNAi silencing. Cell culturing and RNA interference (RNAi) gene silencing were performed as previously described (20). Briefly, the authenticated human ACC cell lines SW-13 and NCI-H295R were purchased from the American Type Cell Collection (Manasas, VA). SW-13 cells were maintained under sterile conditions in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% certified fetal bovine serum and 10,000 U/mL penicillin/streptomycin (Life Technologies) in a standard humidified incubator at 37.0 C
and 5% CO2. NCI-H295R were maintained under sterile conditions in DMEM/F12 supplemented with 5% NuSerum, 0.1% Insulin-Transferrin-Selenium, and 10,000 U/mL penicillin/streptomycin (Life Technologies) in a standard humidified incubator at 37.0 C and 5% CO2.
RNAi gene silencing was carried out with 3 unique 27-mer siRNA duplexes (designated siA, siB, and siC) targeting BCL9 (Human) as previously described (20). Universal scrambled negative control siRNA was used as non-specific control (all from Origene). Briefly, Lipofectamine 2000-mediated transfection was carried out in Opti-MEM according to the manufacturer’s recommendations (ThermoFisher Scientific) in 6-well plates with starting densities of 100,000 cells/well of SW-13 and NCI-H295R. Transfection medium was replaced with regular growth medium after 6 hours of transfection. Cells were lysed for RNA extraction and gene expression analysis at 24 hours post-transfection.
Clonogenic Assays. Five thousand cells per well of SW-13 and NCI-H295R were plated in 6-well plates and allowed to grow for 5-12 days in growth medium at 37.0 C. On day 5 (SW- 13), or 12 (NCI-H295R) cells were washed with PBS, fixed with 3.7% formaldehyde for 20 minutes, and stained with 0.05% crystal violet for 30 minutes. The wells were washed with deionized water until the flow-through water became completely clear, and air-dried. Colonies with 8 +/- 2 cells were counted and averaged from 6 wells.
Statistical Analysis. A 2-tailed t-test was used to assess differences in two groups with continuous variables. For variables with greater than two dependent values, a one-way analysis of variance (ANOVA) was used. Pearson correlation was used to compare matched continuous variables. Survival data were assessed by Kaplan-Meier methods and differences were compared by the Mantel-Cox test. Statistical analyses were performed using GraphPad Prism 7 Software.
RESULTS
Overexpression BCL9 in ACC cells.
Recent studies have implicated a potential role for BCL9 in promoting tumor formation or progression in many tissues. Our targeted WNT-signaling transcription factor expression array analysis on 6 ACC samples identified increased expression of BCL9 in 3 samples of ACC compared to the low levels of expression in normal adrenal cortex (Figure 1A). The increased expression of BCL9 observed in a sub-set of ACCs did not appear to be associated with expression levels of the canonical WNT signal driver, CTNNB1 (Figure 1A). To validate this observation and to test whether the BCL9 dysregulation pattern is shared by benign adrenal tumors as well, we examined mRNA expression patterns of BCL9 in tumor tissues from 17 ACA and 10 ACC patients, in comparison to normal adrenal tissue. As shown in Figure 1B, overall expression levels in ACC were approximately 1.82 times higher compared to normal adrenal tissue (p<0.05), while overall expression in adenomas was only 1.11 times higher compared to normal tissue (Table 1, p<0.05). Messenger RNA expression levels were slightly higher in a few ACA samples tested (Figure 1B), but overall expression levels were not statistically different compared to normal adrenal tissue samples.
Using immunohistochemistry technique, protein expression was also analyzed in representative samples of normal adrenal tissue, as well as ACA and ACC tissues. In normal adrenal tissue, BCL9 expression was spatially specific and was most conspicuous in the zona glomerulosa (ZG). In contrast, minimal protein expression was observed in the other two cortical zones, the zona fasciculata (ZF) and the zona reticularis (ZR) of the histologically normal cortex (Figure 2). ACC tumors samples demonstrated overall higher expression levels compared to
normal and ACA tissue with noticeable diffuse cytoplasmic and nuclear expression patterns (Figure 2). These findings, in conjunction with the mRNA findings, suggest that BCL9 expression is frequently upregulated in ACC.
BCL9 promotes in vitro clonal growth.
olenhandelnden
Previous studies have demonstrated BCL9 to promote in vitro tumor cell growth (15), though this has not been specifically tested in ACC. We used two widely studied ACC cell lines (SW-13 and NCI-H295R) to investigate the potential consequence of BCL9 overexpression in adrenocortical carcinogenesis. Of note, NCI-H295R cells are known to contain a CTNNB1 mutation that causes constitutive activation of ß-Catenin (21). Established RNAi gene silencing techniques were utilized to inhibit BCL9 expression in these cell lines. As shown in Figure 3A, targeted siRNA knockdown of BCL9 expression resulted in the suppression of BCL9 mRNA levels of over 80% and 40% in SW-13 and NCI-H295R cells, respectively, while mock transfection with scrambled siRNA had no significant effect on mRNA transcript levels. Silencing of BCL9 expression significantly reduced SW-13 cells ability to form independent clones in isolation, as demonstrated by the clonogenic growth assay (Figure 3B; left & Figure 3C). The 4-fold decrease in clonogenic growth observed was similar to previous reports in other cancer cell lines (16, 17). In contrast, BCL9 silencing did not interfere with the clonogenic growth potential of NCI-H295R cells (Figure 3B; right & Figure 3C), suggesting that ß-Catenin activation is, in part, possibly sufficient to promote tumor growth in this particular context. Together, these findings suggest that increased expression of BCL9 in ACC tumors promotes neoplastic progression, potentially by activating the Wnt tumorigenic pathway. BCL9 upregulation correlates with the malignant phenotype of adrenocortical tumors.
Previous studies have demonstrated a potential role of BCL9 in tumor invasion and metastases in colon cancers (17, 22). Although limited in cohort size, this study also demonstrated a significant correlation between increased expression of BCL9 and malignant adrenocortical tumors (p<0.05). Only 5.8% of the ACA cohort showed a greater than 2-fold increase in BCL9 expression, while 40% of the ACC samples tested exhibited a 2-fold increase. (Figure 1B). Various clinical parameters (patient gender, age, tumor size, ENSAT stage, metastasis, hormone secretory status, and disease-free survival) were assessed in the study cohort and did not show any significant association with BCL9 expression pattern. Interestingly, there was a trend of higher BCL9 expression levels in older patients, though this did not reach statistical significance (p=0.052).
DISCUSION
Aberrant Wnt/B-Catenin signaling plays a critical role in ACC tumorigenesis. In addition to the well-established role of the central player CTNNB1 in adrenocortical carcinogenesis, recent studies also showed key roles for WNT negative regulators such as DKK3 in adrenocortical malignancy (16). To our knowledge, this is the first study to investigate the potential role of BCL9, a positive regulator of ß-Catenin function, in adrenocortical tumorigenesis. This study shows a significant correlation with increased expression and the malignant status of adrenocortical tumors. The cause of BCL9 overexpression observed in a subset of ACCs here is unknown. Although genetic dysregulation is plausible, recent studies utilizing whole-exome sequencing techniques did not identify BCL9 gene variants in ACC (7, 8). Similarly, studies assessing changes in gene methylation patterns also did not identify BCL9 to be affected significantly (23-25). A more recent comprehensive study also did not identify BCL9
to be specifically affected by point mutations, gene copy alterations, or gene methylation alterations (26). It has been shown, however, that microRNA miR-30a can function as a tumor suppressor and regulate BCL9 function in the setting of gastric cancer (27). Other mechanisms, including transcription factors dysregulation, need to be explored.
In addition to mRNA overexpression, additional mechanisms also may be involved in maintaining higher levels of BCL9 protein expression to facilitate ß-Catenin-promoted oncogenic signaling. BCL9 has been shown to bind ß-Catenin (13) and thus concomitant overexpression of B-Catenin, frequently seen in ACC, may have a synergistic effect on the stability and/or activity of both proteins. Further studies assessing BCL9 expression in relation to B-Catenin expression, function, and constitutive WNT signaling may be elucidating.
Histopathological diagnosis in this rare tumor remains a challenge and additional molecular markers to differentiate malignant from benign tumors is needed. The Ki67 index is the only routinely utilized marker and provides both diagnostic and prognostic information (28). Here we showed that BCL9 gene expression was higher in ACC samples compared to ACA samples, though not all ACC tumors exhibited increased mRNA expression levels and some ACA tumors exhibited increased expression levels as well. It remains unclear whether adrenal adenomas are pre-malignant lesions and it is likely that only small subsets of adenomas possess malignant potential. Whether BCL9 overexpression is unique to adrenal cancer formation or occurs in a broader context of adrenal neoplasia, remains to be determined. Nevertheless, BCL9 overexpression could serve a molecular marker of malignant potential, but further analysis with a larger cohort and also with combinations of other molecular markers, would be needed for validation.
Despite recent advances in our understanding of the genetic and epigenetic causes of adrenocortical carcinogenesis, progress in improving treatment outcomes for adjuvant and salvage therapy has remained limited. In recent years, multiple targeted regimens, including the use of tyrosine kinase, mTOR, and IGF-1R inhibitors, have been introduced but demonstrated lackluster efficacy (1). Targeting BCL9 in conjunction with ß-Catenin may prove to be a promising route, as has been previously suggested, especially in the context of the WNT-targeted pharmaceuticals that are currently in clinical trial for a variety of caner types (13, 29). Although the mutational landscape of ACC has been proven to be very heterogeneous, targeting WNT pathway through its regulatory components such as BCL9 still remains a very attractive target for a significant proportion of ACCs for an improved clinical outcome.
CONCLUSION
This study demonstrates for the first time that BCL9 is overexpressed in malignant adrenocortical tumors and promotes ACC colony cell growth. These findings suggest that BCL9 overexpression may serve as an alternative driver of Wnt/B-Catenin activation in ACC and could represent a potential molecular and diagnostic marker of adrenocortical tumor malignancy.
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ACCEPTED MANUS CRIPT
| Characteristic | ACA, n (%) | ACC, n (%) | p Value |
|---|---|---|---|
| Tumor type | 17 | 10 | 0.011 |
| Sex | 0.772 | ||
| Male | 3 (17.6) | 2 (20.0) | |
| Female | 14 (82.4) | 8 (80.0) | |
| Age, y, mean ± SD | 52.8 ±10.2 | 57.1 ±11.7 | 0.052 |
| Tumor size, cm | 0.224 | ||
| Mean ± SD | 4.0± 1.6 | 10.0±3.3 | |
| Range | 1.5-6.5 | 5.5-14.0 | |
| ENSAT 2008 stage | 0.069 | ||
| I | NA | 0 (0.0) | |
| II | NA | 1 (10.0) | |
| III | NA | 6 (60.0) | |
| IV | NA | 3 (30.0) | |
| Hormone production | 0.550 | ||
| Aldosterone | 2 (11.8) | 0 (0.0) | |
| Cortisol | 3 (17.6) | 3 (30.0) | |
| Androgen | 0 (0.0) | 3 (30.0) | |
| Multi-secreting* | 0 (0.0) | 2 (20.0) | |
| Nonfunctional | 9 (53.0) | 2 (20.0) | |
| No information available | 3 (17.6) | 0 (0.0) | |
| Outcome | 0.499 | ||
| Alive, no recurrence | 14 (82.4) | 2 (20.0) | |
| Alive, recurrent | 0 (0.0) | 3 (30.0) | |
| Death from disease | 0 (0.0) | 4 (40.0) | |
| Death from other causes | 0 (0.0) | 1 (10.0) | |
| Lost to follow-up | 3 (17.6) | 0 (0.0) |
*Tumors secreting 2 or more of the following hormones: aldosterone, cortisol, testosterone, or dehydroepiandrosterone.
ACA, adrenocortical adenoma; ACA, adrenocortical carcinoma; ENSAT, European Network for the Study of Adrenal Tumors; NA, not applicable.
Figure Legends
Figure 1. BCL9 gene expression analysis. (A) Transcription factor expression analysis was performed using the RT2 Profile PCR Array Human WNT Signaling Pathway (Qiagen). B-Cell CLL/Lymphoma 9 (BCL9) was found to be overexpressed in 3 of 6 samples of adrenocortical carcinoma (ACC) tested (T1, T2, & T4). Shades of red and brown signify increased expression; shades of green signify decreased expression. T, ACC; N, normal. (B) Relative messenger RNA expression levels in adrenocortical adenoma (ACA) samples (n=17) and ACC samples (n=10) were measured by real-time quantitative polymerase chain reaction and compared with expression levels in normal adrenal tissues (n=16). Expression levels were higher in ACC samples compared to ACA and normal samples. Error bars, SEM; * , p=0.006.
Figure 2. BCL9 immunohistochemical analysis. Immunostaining of B-Cell CLL/Lymphoma 9 (BCL9) in representative samples of adrenocortical carcinoma (ACC) (right) demonstrated increased protein expression compared to adrenocortical adenoma (ACA) (middle) and normal (left) tissue samples. Original magnification, X400; BCL9, brown; Hematoxylin, blue.
Figure 3. In vitro analysis. (A) B-Cell CLL/Lymphoma 9 (BCL9) expression was inhibited by small interfering RNA (siRNA) transfection in SW-13 (left) and NCI-H295R (right) cells. Bars, mean; error bars, SEM; * , p<0.004. (B) After 5 (SW-13, top) or 12 (NCI-H295R, bottom) days of colony growth, cells were washed with PBS, fixed with 3.7% formaldehyde, washed with PBS, and stained with 0.05% crystal violet. Colonies with 8 +/- 2 cells were counted by higher power field. Bars, mean; error bars, SEM; * , p<0.0001. (C) Representative photomicrographs of SW-13 cells in Figure 3B, are shown.
Precis
This is the first study to identify BCL9 gene amplifications in adrenocortical carcinoma (ACC) and demonstrates BLC9 to promote cancer cell growth. These findings suggest that BCL9 may serve as a novel diagnostic marker and potential therapeutic target in ACC treatment.
ACCEPTED MANUS CRIPT
V
V
V
BCL9
CTNNB1
T1
T2
T3
T4
T5
T6
N1
N2
N3
A
4
*
A
Relative Gene Expression
3-
.
4
2-
1-
Â
0
.
B
Normal (n=16)
ACA (n=17)
ACC (n=10)
ACCEPTED M
Normal
ACA
ACC
ACCEPTED MANU
1000
5000
400
Relative BCL9 Gene Expxpression
Number of Colonies Per Well
800
Number of Colonies Per Well
4000
600
3000
2000
1.0-
200
1000
0
0
Scramble SİRNA
BCL9 SİRNA
Scramble SİRNA
BCL9 SİRNA
Lipo
Lipo
0.5
B
SW-13
NCI-H295R
0.0
Normal Scramble SİRNA SW-13
BCL9 SIRNA
A
Normal Scramble BCL9 SIRNA SİRNA
NCI-H295R
C
Normal Control
Scrambled SİRNA
BCL9 siRNA
ACCEPTED
ACCEPTED MANUSCRIPT
AUTHOR CONTRIBUTIONS FORM
Individuals claiming authorship should meet all 3 of the following conditions in accordance with the “Consensus Statement on Surgery Journals Authorship-2005”:
1) Authors make substantial contributions to conception and design, and/or acquisition of data, and/or analysis and interpretation of data;
2) Authors participate in drafting the article or revising it critically for important intellectual content; and
3) Authors give final approval of the version to be submitted and any revised version.
Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Allowing one’s name to appear as an author without having contributed significantly to the study or adding the name of an individual who has not contributed or who has not agreed to the work in its current form is considered a breach of appropriate authorship.
Ghost-writing is NOT acceptable. No one, other than the authors listed below, should have contributed substantially to the writing and revising of the manuscript. Contributors who do not meet the criteria for authorship should be listed in the acknowledgment. Examples include: individuals who allowed their clinical experience to be included, a person who provided purely technical help, copyediting, proofreading or translation assistance (NO ghostwriters allowed), or a department Chair who provided only general support.
Groups of persons who have contributed materially to the paper, but whose contributions do not justify authorship may be listed under a heading such as “clinical investigators” or “participating investigators,” and their function or contribution should be described; for example, “served as scientific advisors,” “critically reviewed the study proposal.“] If you have any question about this, contact that editorial office before submitting your manuscript at jacsedit@facs.org or 312-202-5316.
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Please type each author’s LAST NAME ONLY next to the appropriate category.
Study conception and design: Brown, Nicolson, Korah, Carling.
Acquisition of data: Brown, Nicolson, Korah.
Analysis and interpretation of data: Brown, Nicolson, Korah, Carling.
Drafting of manuscript: Brown, Nicolson, Korah, Carling.
Critical revision: Brown, Nicolson, Korah, Carling.