Overexpression of Rabl3 and Cullin7 is associated with pathogenesis and poor prognosis in hepatocellular carcinoma

    Overexpression of Rabl3 and Cullin7 is associated with pathogenesis and poor prognosis in hepatocellular carcinoma Jun An MD, PhD, Zhiyong Liu MD, Qiong Liang MD, Yuhang Pan MD, Haifeng Li MD, Ruizhi Wang MD, Yi Jin MD PII: DOI: Reference:

S0046-8177(17)30258-7 doi: 10.1016/j.humpath.2017.07.008 YHUPA 4283

To appear in:

Human Pathology

Received date: Revised date: Accepted date:

14 March 2017 27 June 2017 12 July 2017

Please cite this article as: An Jun, Liu Zhiyong, Liang Qiong, Pan Yuhang, Li Haifeng, Wang Ruizhi, Jin Yi, Overexpression of Rabl3 and Cullin7 is associated with pathogenesis and poor prognosis in hepatocellular carcinoma, Human Pathology (2017), doi: 10.1016/j.humpath.2017.07.008

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ACCEPTED MANUSCRIPT Overexpression of Rabl3 and Cullin7 is associated with pathogenesis and poor prognosis in hepatocellular carcinoma

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Running title

Overexpression of Rabl3 and Cullin7 in hepatocellular carcinoma

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Authors’ full names

Jun An, MD, PhD1&, Zhiyong Liu, MD2&, Qiong Liang, MD3, Yuhang

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Pan, MD3, Haifeng Li, MD3, Ruizhi Wang, MD4, Yi Jin, MD3 * Department of Cardiothoracic Surgery, the third Affiliated Hospital of

Sun Yat-Sen University, Guangzhou, China

Department of Emergency Medicine, the Third Affiliated Hospital, Sun

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Yat-Sen University, Guangzhou, China Department of Pathology, Guangdong Provincial Key Laboratory of

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Liver Disease Research, the Third Affiliated Hospital, Sun Yat-Sen

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University, Guangzhou, China Department of Clinical Laboratory, the First Affiliated Hospital, Sun

Yat-Sen University, Guangzhou, China Author contributions Yi Jin designed the research; Jun An, Zhiyong Liu, and Qiong Liang performed the research; Haifeng Li and Ruizhi Wang analyzed the data; Yuhang Pan and Zhiyong Liu wrote the manuscript. The authors declare that they have no conflict of interest. &

Equal contributors 1

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Corresponding author:

Yi Jin, MD

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Department of Pathology, Guangdong Provincial Key Laboratory of Liver Disease Research, the Third Affiliated Hospital, Sun Yat-Sen University,

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Guangzhou, China

China E-mail: [email protected] Tel.: +86-20-85253436

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Fax: +86-20-85253436

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Mailing address: 600 Tianhe Road, Guangzhou, Guangdong, 510630,

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Running head: Overexpression of Rabl3 and Cullin7 in HCC.

Word count (main text): 2581 wards

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ACCEPTED MANUSCRIPT Abstract The expression of Rabl3 and Cullin7 is relevant to the carcinogenesis of

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certain cancers. However, the relationship of this expression with hepatocellular carcinoma remains unclear. To study the protein expression

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of Rabl3 and Cullin7 and to evaluate their role in hepatocarcinogenesis, in 162 cases of hepatocellular carcinoma, we used immunohistochemistry

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to investigate the expression of Rabl3 and Cullin7 in both the cancer tissues and the normal hepatic tissues around the hepatocellular carcinoma. The results demonstrated that the rates of positive Rabl3 and

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Cullin7 expression were 80.2% and 69.1%, respectively, in hepatocellular

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carcinoma tissues. However, the rates of positive Rabl3 and Cullin7 expression were 31.5% and 29.0%, respectively, in adjacent normal

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hepatic tissues. Rabl3 and Cullin7 were expressed at significantly higher

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rates in hepatocellular carcinoma compared with adjacent normal hepatic tissues (P<0.01). The rates of positive Rabl3 and Cullin7 expression were higher in the hepatocellular carcinoma tissues of patients with lymph node metastasis, tumor thrombi in the portal vein and an advanced clinical stage (P<0.05). A positive correlation between the expression of Rabl3 and the expression of Cullin7 (r=0.27, P<0.001) was also observed in our hepatocellular carcinoma cohort. Moreover, patients with positive expression for both Rabl3 and Cullin7 had a remarkably shorter survival time compared with patients with negative expression for both proteins 3

ACCEPTED MANUSCRIPT (P<0.05). Therefore, the expression of the Rabl3 and Cullin7 proteins may play an important role in the pathogenesis and progression of

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hepatocellular carcinoma and could be used as a prognostic indicator in

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patients with hepatocellular carcinoma.

Keywords: Hepatocellular carcinoma; Immunohistochemistry; Rabl3;

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Cullin7; Protein; Pathogenesis

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ACCEPTED MANUSCRIPT 1. Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent

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malignancies worldwide, and its morbidity and mortality rates have escalated in recent years [1-3]. At present, the incidence of HCC is

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increasing, and the disease is expected to become the third leading cause of cancer-associated mortality in the world [4, 5]. At the time of being

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diagnosed, the majority of patients have already entered the middle-advanced to late stages, and the 5-year survival rate is extremely low [6-8]. In recent years, some progress has been made in elucidating

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the pathogenesis of hepatocellular carcinoma [9, 10]; however, no

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particular molecular marker has been reported for HCC diagnosis [11]. Meanwhile, HCC progression can be partly predicted by evaluating the

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[12].

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proliferative activity and assessing microvascular invasion of HCC cells

Assessment of the prognosis of HCC still relies on certain pathological characteristics, such as tumor size, grade, and stage and lymph node metastasis [13]. However, these traditional clinicopathological parameters only offer limited information for prognosis prediction [14, 15]. Thus, it is very important to further clarify the molecular mechanisms of hepatocarcinogenesis and to identify key new molecular markers for diagnosing and assessing the prognosis of HCC. The Rabl3 gene is a member of the Rab subfamily, which possesses 5

ACCEPTED MANUSCRIPT intrinsic GTPase activity to control GDP/GTP conversion. It has been shown that there is a relationship between alterations in the Rab small

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GTPases and oncogenesis [16-20]. For example, Li et al. found that Rabl3 expression was related to paclitaxel sensitivity in breast cancer

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cells. Their results specifically demonstrated that overexpression of Rabl3 could prevent tumor cells from undergoing paclitaxel-induced apoptosis

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and consequently increase resistance to paclitaxel. Consistent with this finding, silencing of Rabl3 expression could increase paclitaxel-induced apoptosis in MDA-MB-231 cells [18]. As a result, overexpression of

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Rabl3 may improve proliferation and inhibit apoptosis in human cancer

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cell lines.

Hepatocarcinogenesis is a multi-gene and multi-stage disease process

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that likely involves various pathogenic factors, such as Cullin7. Cullin7

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assembles an SCF-like E3 complex containing Skp1, the Fbw8 F-box protein, and ROC1 and may interact with the Skp1-Fbw8 heterodimer [21-23]. Recently, Men and colleagues revealed that Cullin7 expression is increased in human primary lung cancer tissues and that its aberrant expression may contribute to the pathogenesis of lung cancer [24], thus providing evidence that Cullin7 functions as a novel oncogene in lung cancer. In another study, the expression of Cullin7 mRNA was significantly higher in epithelial ovarian cancer (EOC) compared with normal ovarian surface tissues [25]. These results suggest that Cullin7 6

ACCEPTED MANUSCRIPT may serve as an indicator of poor prognosis in patients with EOC. Taken together, the above research results powerfully suggest that Cullin7 may

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play an important role in carcinogenesis and cancer progression. It has been reported that Rabl3 and Cullin7 are FAK/ERK signaling

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pathway molecules that may affect each other during carcinogenesis and development [18, 21]. However, the co-expression dynamics of Rabl3

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and Cullin7 in HCC and their roles in hepatocarcinogenesis have not been elucidated. Whether the co-expression of these proteins is associated with the pathogenesis and progression of HCC has also not been reported. In

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the present study, we utilized immunohistochemistry to investigate the

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expression of Rabl3 and Cullin7 in HCC of various clinicopathological grades. We found that the expression of Rabl3 and Cullin7 was higher in

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cancer tissues than in adjacent normal hepatic tissues and was correlated

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with the clinical staging, lymph node metastasis and cancer thrombi in the portal vein in HCC. Moreover, patients with positive expression for both Rabl3 and Cullin7 had significantly shorter survival compared with patients with negative protein expression. This study thus suggests that the expression of Rabl3 and Cullin7 may provide novel diagnostic and prognostic insights and may serve as a new therapeutic target in HCC.

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ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1.

Ethics statement

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All procedures performed on human participants were in accordance with the ethical standards of the institutional and/or national research

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committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved

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by the Clinical Research Ethics Committee of the Third Affiliated Hospital, Sun Yat-sen University. Written informed consent was obtained from all the participants, and the ethical guidelines of the Helsinki

Clinical specimens

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2.2.

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Declaration were followed.

Specimens were obtained from the archives of the Department of

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Pathology of the Third Affiliated Hospital, Sun Yat-sen University; these

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specimens were specifically paraffin-embedded tissue sections from 162 patients with HCC who did not have a history of chemotherapy or radiotherapy that were collected between January 1, 2008, and December 31, 2009. Additionally, a total of 162 normal liver tissue specimens that were obtained 2cm from the periphery of the cancer site were utilized as controls. The histopathological diagnosis of all specimens was verified by a trained pathologist. Among the 162 patients (144 men and 18 women), with a median age of 53 years (range 19-72 years), there were 37 cases of well-differentiated carcinoma, 112 cases of moderately differentiated 8

ACCEPTED MANUSCRIPT carcinoma, and 13 cases of poorly differentiated carcinoma. According to the TNM system of the American Joint Committee on Cancer (AJCC)

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[26], 64 cases were classified as stage A, 11 cases were stage B, 67 cases were stage C, and 20 cases were stage D.

Overexpression of Rabl3 and Cullin7 proteins in HCC

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2.3.

The expression of Rabl3 and Cullin7 in HCC tissues was examined by

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immunohistochemistry using the NovolinkTM Polymer Detection Systems. The thickness of the tissue section for immunohistochemistry was 4 µm. All sections were routinely deparaffinized and rehydrated, rinsed in

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phosphate-buffered saline (PBS, pH 7.4) and then treated for antigen

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retrieval. Sections were specifically treated by heating in Tris-EDTA buffer at pH 8.0 in an autoclave sterilizer for 2 min. After the sections

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cooled to room temperature, they were subsequently rinsed in PBS and

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immersed in 3% H2O2 for 5 min to block the endogenous enzymes. To block non-specific antibody binding, the sections were incubated with normal goat serum at 37°C for 5 min. Afterward, labeling was performed with a rabbit anti-human monoclonal Rabl3 antibody (dilution 1:150, Abcam) and polyclonal Cullin7 antibody (dilution 1:150, Abcam). After washing in PBS, the sections were incubated with Novolink™ Polymer for 15 min. Following further rinsing in PBS, the protein expression was visualized by incubation with 3,3’-diaminobenzidine and counterstaining with hematoxylin. PBS substituted for the primary antibody was used as a 9

ACCEPTED MANUSCRIPT negative control. Finally, the sections were dehydrated, rendered transparent, covered with coverslips and sealed with neutral gum.

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Assessment of Rabl3 and Cullin7 overexpression in HCC

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2.4.

patients

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The specimens were assessed by two experienced pathologists. Rabl3 expression was localized predominantly in the cytoplasm, and Cullin7

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expression was localized mainly in the nucleus. Sections were considered positive if expression was present in more than 10% of the cells in HCC tissues or normal hepatic tissues around HCC tissues [27]. Statistical analysis

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2.5.

The data were analyzed using SPSS version 13.0. (Chicago, IL, USA).

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The χ2 test was used to evaluate the statistical associations between the

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expression of Rabl3 and Cullin7 and the clinicopathological parameters.

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In addition, the correlation between two variables was evaluated using the Spearman rank correlation test. A value of P<0.05 was considered statistically significant.

3. Results 3.1 Association between Rabl3 and Cullin7 protein expression and clinicopathological characteristics of patients with HCC Immunohistochemical detection indicated that the protein expression of Rabl3 was predominantly in the cytoplasm (Fig. 1a, 1b). Rabl3 10

ACCEPTED MANUSCRIPT expression was positive in 80.2% (130/162) of HCC specimens, compared with 31.5% (51/162) of normal hepatic tissue specimens.

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Statistical analysis demonstrated that there was a significant difference in Rabl3 expression between HCC and adjacent normal hepatic tissues

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(P<0.01) (Table 1). Rabl3 protein expression was associated with cancer thrombi in the portal vein, lymph node metastasis and clinical staging in (P<0.05),

whereas

it

was

not

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HCC

associated

with

other

clinicopathological characteristics (P>0.05) (Table 2). The protein expression of Cullin7 was predominantly present in the

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nucleus (Fig. 1c, 1d). The rate of positive Cullin7 expression was 69.1%

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in HCC specimen tissues, whereas it was 29.0% in normal hepatic tissues adjacent to cancer tissues. Statistical analysis also demonstrated that there

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was a significant difference in the expression of Cullin7 between HCC

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and normal hepatic tissues (69.1% vs 29.0%) (P<0.01) (Table 1). Furthermore, the protein expression of Cullin7 was related to cancer thrombi in the portal vein, lymph node metastasis and clinical staging in HCC (P<0.05), whereas it was not related to other clinicopathological characteristics (P>0.05) (Table 2). In the present research, the correlation between the protein expression of Rabl3 and Cullin7 and HCC was further evaluated. The samples with positive expression for both Rabl3 and Cullin7 was 98, and the negative expression for both Rabl3 and Cullin7 was 18. Statistical analysis 11

ACCEPTED MANUSCRIPT revealed that there was a remarkable correlation between the expression of Rabl3 and the expression of Cullin7. In particular, Spearman rank

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correlation analysis showed a positive correlation between Rabl3 and Cullin7 protein expression in HCC specimens (r=0.27, P<0.001).

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Therefore, the higher the Rabl3 expression was, the higher the expression of Cullin7 was (Table 3).

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3.2 Univariate and multivariate survival analyses In HCC tissues, the 1- and 3-year survival rates for patients in whom positive protein expression was observed for both Rabl3 and Cullin7 were

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66.3% (65/98) and 30.6% (30/98), respectively. In comparison, the 1- and

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3-year survival rates for patients with negative expression for Rabl3 and Cullin7 were 83.3% (15/18) and 61.1% (11/18), respectively. Furthermore,

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the 1- and 3-year survival rates were respectively 75.0% (24/32) and

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46.9% (15/32) for patients expressing Rabl3 alone and 78.6% (11/14) and 50.0% (7/14) for patients expressing Cullin7 alone. Thus, the 1- and 3-year survival rates were lower for patients with positive expression for both Rabl3 and Cullin7 than for those with negative expression for both (P = 0.03) (Figure 2).

4. Discussion Rab is a large subfamily of small GTPases that play an important role in multiple processes regulating cellular transport and the formation of the 12

ACCEPTED MANUSCRIPT cytoskeleton. It has been reported that Rabs could improve the proliferation and invasiveness of tumor cells. In particular, Rabl3 is a

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member of the Rab group of proteins that is overexpressed in tumor cells and that is related to enhanced cell proliferation and reduced cell death,

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and it also has a close association with the progression and aggressiveness of certain kinds of cancer [16-18]. Recently, investigations

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have also shown that overexpression of Rabl3 could prevent tumor cells from undergoing apoptosis and could enhance the proliferation, invasion and metastasis of tumor cells [17].

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In the present research, we observed that Rabl3 expression was present

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in 80.2% of HCC tissues but in fewer adjacent normal hepatic tissues (31.5%, P<0.01), supporting the concept that Rabl3 may play an

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important role in the pathogenesis and progression of HCC. It has also

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been reported that Rabl3 is a component of the FAK/ERK signaling pathway and can stimulate the differentiation and proliferation of tumor cells by activating signaling pathways [17]. Therefore, Rabl3 may play an important role in hepatocarcinogenesis. Furthermore, in the present study, statistical analysis showed that overexpression of Rabl3 was associated with portal vein tumor thrombi, lymph node metastasis and clinical staging. In contrast, Rabl3 expression was unrelated to patient gender or age, the number of tumors, tumor grade, liver function, or other clinicopathological parameters. Finally, the rate of Rabl3 expression was 13

ACCEPTED MANUSCRIPT higher in HCC specimens from patients with portal vein tumor thrombi, lymph node metastasis and HCC of clinical stage C or D. Taken together,

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the findings suggest that overexpression of Rabl3 may not only directly stimulate tumor cell proliferation and enhance carcinogenesis but also

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improve the migration of cancer cells and enhance tumor growth and progression. The high expression of the Rabl3 protein in HCC may be

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related to the activity of certain transcription factors. One such factor is Cullin7, a crucial regulator of gene transcription that is intimately correlated with carcinogenesis [27]. In the present research, Cullin7 was

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expressed in 69.1% of HCC tissues, whereas expression was observed in

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only 29.0% of adjacent normal hepatic tissues. The expression of Cullin7 was thus significantly higher in HCC than in adjacent normal hepatic

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tissues (P<0.01), suggesting that Cullin7 may also participate in the

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pathogenesis of HCC. Additionally, statistical analysis showed that the expression of Cullin7 was remarkably higher in patients with HCC of clinical stage C or D, tumor thrombi in the portal vein and lymph node metastasis than in patients with HCC of clinical stage A or B, tumors without lymph node metastasis and no tumor thrombi in the portal vein. In this study, statistical analysis illustrated that the expression of Rabl3 was linearly and positively associated with the expression of Cullin7 in HCC. In other words, a higher level of Rabl3 was associated with a higher level of Cullin7. Li et al. confirmed that Rabl3-related signaling 14

ACCEPTED MANUSCRIPT proteins play important roles in cellular proliferation and migration and posited that Rabl3 is a component of the FAK/ERK signaling pathway

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[17]. However, Cullin7 is a crucial transcription factor that can inhibit apoptosis, activating the FAK/ERK signaling pathway by regulating p53

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[20]. It was reported that Cullin7 activates FAK/ERK signaling pathways and also contributes to overexpression of Rabl3. Thus, overexpression of

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Rabl3 and Cullin7 can have a synergistic effect in facilitating the pathogenesis of HCC and regulating the migration of HCC cells. Moreover, the 1- and 3-year survival rates of HCC patients with

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positive expression for both the Rabl3 and the Cullin7 proteins were

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lower than those of patients with negative expression for both proteins. It is possible that co-expression of Rabl3 and Cullin7 affected the prognosis

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of patients with HCC, with HCC patients with co-expression of Rabl3

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and Cullin7 having a poor prognosis. In short, the protein expression of Rabl3 and Cullin7 was remarkably higher in HCC than in adjacent normal hepatic tissues and probably crucially contributes to the carcinogenesis and progression of HCC. The expression of Rabl3 and Cullin7 may thus be utilized as a diagnostic and prognostic marker of HCC.

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ACCEPTED MANUSCRIPT Acknowledgments This study was funded by the Science and Technology Program of

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Guangdong Province (No. 2014A020212155).

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Conflict of Interest

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All the authors declare that they have no conflict of interest.

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Positive Rabl3 and Cullin7 protein expression in

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hepatocellular carcinoma (HCC).

(a & b) Rabl3 immunohistochemical staining. Rabl3 expression was

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predominantly observed in the cytoplasm and was visualized as brown-yellow staining in HCC. (c & d) Cullin7 immunohistochemical

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staining. Expression of Cullin7 was observed predominantly in the nucleus and was visualized as brown-yellow staining in HCC (c: 20×10, d:

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40×10).

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Figure 2. Comparison of the survival times of patients with HCC. Comparison of the survival times of patients with HCC depending on

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Rabl3 and Cullin7 expression. The 1- and 3-year survival rates were

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lower for patients with positive Rabl3 and Cullin7 protein expression than for patients with negative expression for both proteins (P=0.03).

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Figure 1

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Figure 2

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ACCEPTED MANUSCRIPT Table 1. The expression of Rabl3 and Cullin7 between HCC and adjacent normal tissue Rabl3

Cullin7

negative positive (%) 162

32

130 (80.2)

Normal

162

111

51(31.5)

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HCC

negative positive (%)

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n

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Groups

50

112(69.1)

115

47 (29.0)

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There was a significant difference in the expression of Rabl3 and Cullin7 between HCC and adjacent normal hepatic tissue (P< 0.01).

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ACCEPTED MANUSCRIPT Table 2 Relationship between the expression of Rabl3 and Cullin7 and clinico-pathological parameters in HCC n

Rabl3

Cullin7

negative(%)

positive(%)

P

negative(%)

positive(%)

P

0.364

T

Parameter

44（30.6）

100（69.4）

0.810

6（33.3）

12（66.7）

144

27（18.8）

117（81.2）

Female

18

5（27.8）

13（72.2）

＜50

97

21（21.6）

76（78.4）

≥50

65

11（16.9）

54（83.1）

＜5

90

17（18.9）

73（81.1）

≥5

72

15（20.8）

57（79.2）

Single

116

21（18.1）

95（81.9）

multitude

46

11（23.9）

35（76.1）

＜400

98

17（17.3）

81（82.7）

≥400

64

15（23.4）

49（76.6）

have

69

6（8.7）

63（91.3）

no

93

26（28.0）

67（72.0）

have

33

2（9.9）

31（93.9）

no

129

30（23.3）

99（76.7）

37

11（33.9）

26（70.3）

112

18（29.1）

13

Child A

Age( years) 0.459

RI P

Male

AC

Sex

65（67.0）

18（27.7）

47（72.3）

29（32.2）

61（67.8）

21（29.2）

51（70.8）

34（29.3）

82（70.7）

16（34.8）

30（65.2）

31（31.6）

67（68.4）

19（29.7）

45（70.3）

11（15.9）

58（84.1）

39（41.9）

54（58.1）

5（15.2）

28（84.8）

45（34.9）

84（65.1）

12（32.4）

25（67.6）

94（83.9）

33（29.5）

79（70.5）

3（30.6）

10（76.9）

5（38.5）

8（61.5）

106

21（19.8）

85（80.2）

34（32.1）

72（67.9）

Child B

36

6（16.7）

30（83.3）

11（30.6）

25（69.4）

Child C

20

5（25.0）

15（75.0）

5（25.0）

15（75.0）

A

64

21（32.8）

43（67.2）

30（46.9）

34（53.1）

B

11

4（36.4）

7（63.6）

5（45.5）

6（54.5）

C

67

5（7.5）

62（92.5）

12（17.9）

55（82.1）

D

20

2（10.0）

18（90.0）

3（15.0）

17（85.0）

Tumor numbers

ED

AFP(µg/L)

CE

Lymph metastasis

Pathological grade high moderately low

PT

Tumor thrombus

0.757

MA NU

Tumor size（cm）

SC

32（33.0）

0.402

0.341

0.002

0.027

0.185

0.474

0.676

0.497

0.793

0.000

0.029

0.780

Hepatic function 0.754

0.820

Clinical stage

23

0.001

0.001

ACCEPTED MANUSCRIPT Table 3 Relative protein expression of Rabl3 and Cullin7 in HCC Cullin7 negative

98

32

positive negative

14

18

total

112

50

total 130

T

positive

32

RI P

Rabl3

162

AC

CE

PT

ED

MA NU

SC

There is a relationship between the expression of Rabl3 and Cullin7 in HCC (P<0.001，x2=12.04), correlation coefficient, r=0.27.

24

ACCEPTED MANUSCRIPT Highlights Rabl3 expression was significant difference between HCC and normal hepatic tissue.

RI P

T

Cullin7 expression was significant difference between HCC and normal hepatic tissue.

AC

CE

PT

ED

MA NU

SC

Expression of Rabl3 and Cullin7 may be a diagnostic and prognostic marker of HCC.

25