p66Shc promotes HCC progression in the tumor microenvironment via STAT3 signaling

Experimental Cell Research 383 (2019) 111550

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p66Shc promotes HCC progression in the tumor microenvironment via STAT3 signaling

T

Peixin Huanga,b,1, Xuemei Fengc,1, Zhiying Zhaoa,b, Biwei Yanga,b, Tingting Fanga,b, Mengzhou Guoa,b, Jinglin Xiaa,b,* a

Department of Hepatic Oncology, Zhongshan Hospital, Fudan University, Shanghai, 200032, China Liver Cancer Institute & Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai, 200032, China c Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: p66Shc Hepatocellular carcinoma Tumor microenvironment STAT3

The development of hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species. However, the importance and biologic functions of p66Shc in HCC are unclear. The clinical significance of p66Shc was assessed in a large cohort of patients with HCC. High Shc1 expression was closely correlated with poor clinical outcomes and early recurrence of HCC. p66Shc expression was also determined in HCC samples and cell lines and found to be increased. Moreover, knockdown of p66Shc significantly inhibited cell proliferation, motility in vitro and tumor growth in vivo and could attenuate the proliferation, and motility of cells stimulated by activated macrophage conditioned media. Mechanically, p66Shc knockdown inhibited phosphorylation of STAT3 on serine 727 in vitro and in vivo. Our results show that high p66Shc expression in HCC predicts a worse prognosis for survival. Furthermore, p66Shc may serve as a novel candidate target for HCC therapy.

1. Introduction Hepatocellular carcinoma (HCC) is one of the most frequently occurring types of cancer worldwide [1] and has a high rate of associated mortality [2]. The occult nature of HCC can make it difficult to diagnose in the early stages and, as recurrence and metastasis are common, the long-term survival rate of patients with HCC remains low [3–6]. Chronic inflammation contributes to the development of HCC [7] and understanding its contribution to the development of the disease may provide an important route for the discovery of prognostic indicators and potential therapeutic targets. It has long been recognized that chronic inflammation is an essential player in the pathway to tumorigenesis [8] and can affect every facet of tumor development from initiation and progression to metastasis [9]. Chronic inflammation contributes to tumorigenesis by inducing DNA damage; it has a role in tumor progression through its promotion of cell proliferation and resistance to apoptosis, and can also stimulate tissue remodeling and angiogenesis [3,5,6]. Chronic inflammation can induce

inappropriate activation of various transcription factors, including signal transducer and activator of transcription (STAT) family members and nuclear factor-kappa B (NF-κB) [9–12]. These transcription factors in turn function to induce genes involved in pro-tumorigenic processes such as cell proliferation, survival, angiogenesis, and invasion [9]. The induction of transcription factors such as STAT3 and NF-κB is affected by signaling molecules found in the tumor microenvironment (TME). The TME is enriched in immune cells including tumor-associated macrophages and lymphocytes that produce a pro-tumorigenic environment through their release of inflammatory mediators such as cytokines, growth factors, and reactive oxygen species (ROS) [13]. These inflammatory mediators have been implicated in the pathway of malignant transformation through their tumor promoting effects on cancer cell proliferation, survival, motility, and invasion [13]. The SHC gene regulates ROS levels, apoptosis induction, and lifespan in mammals [14]. The Shc (Src homology 2 domain containing) protein is a member of an adaptor family of proteins and can be found in three isoforms based on molecular weight (p46, p52, and p66) [15].

* Corresponding author. Liver Cancer Institute & Zhongshan Hospital, Shanghai medical college of Fudan University, No. 180 Fenglin Road, Shanghai, 200032, China. E-mail addresses: [email protected] (P. Huang), [email protected] (X. Feng), [email protected] (Z. Zhao), [email protected] (B. Yang), [email protected] (T. Fang), [email protected] (M. Guo), [email protected] (J. Xia). 1 Peixin Huang and Xuemei Feng contributed equally to this work.

https://doi.org/10.1016/j.yexcr.2019.111550 Received 29 March 2019; Received in revised form 5 August 2019; Accepted 6 August 2019 Available online 06 August 2019 0014-4827/ © 2019 Elsevier Inc. All rights reserved.

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following treatment with hydrogen peroxide. After blocking, the sections were incubated overnight at 4 °C with Shc1 (1:300; Abcam, MA, USA) the primary antibody. The sections were further incubated with HRP-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) and visualized using 3, 3'-diaminobenzidine (Zhonghshan Golden Bridge Biotechnology, Beijing, China). Counterstaining was performed using hematoxylin. The percentage of immunostaining and the staining intensity (0, negative, 1+, weak; 2+, moderate; and 3+, strong) were documented. An H-score was calculated using the formula: H-score = (% of cells of weak intensity × 1) + (% of cells of moderate intensity × 2) + (% of cells of strong intensity × 3). The maximum H-score would be 300, corresponding to 100% of cells with strong intensity. Shc1 expression was categorized as high or low using the median Shc1 H-score. Samples were scored as either having a low-Shc1 H-score ≤ 89.23 or a HighShc1 H-score > 89.23. Immunofluorescence was performed by dewaxing the sections in xylene followed by rehydration in ethanol. The sections were then incubated for 10 min at 120 °C to facilitate antigen retrieval. The sections were then treated with normal goat serum for 30 min in order to reduce nonspecific binding, followed by incubation overnight at 4 °C with antiShc1 antibody (Abcam, 1:200). Secondary antibodies conjugated with Alexa Flour 488 (Invitrogen) were added for an additional 1 h of incubation. Counterstaining with DAPI (Sigma-Aldrich, St. Louis, MO, USA) was carried out to detect cell nuclei. Images were obtained using a fluorescent microscope (Olympus, Tokyo, Japan).

The longest isoform, p66Shc, has an additional CH2 domain containing a S36 residue which is phosphorylated in response to oxidative stress and has a role in apoptosis [16,17]. While p46Shc and p52Shc are universally expressed, p66Shc is expressed at different levels in different tissues [15]. p66Shc is part of a signal transduction pathway that is activated in response to increased ROS [18] and was initially recognized as a pro-apoptotic protein as its expression has been found to be required for apoptosis initiation in response to oxidative stress and its absence results in abrogation of apoptosis [15]. Furthermore, p66Shc has been found to be a downstream effector of the tumor suppressor gene p53 [19] and in a more recent study was found to act as a tumor suppressor [20]. However, a contradictory role for p66Shc has come to light with the recognition that p66Shc also functions as a proto-oncogene [15]. p66Shc has been shown to have a role in cell proliferation [21,22] and its induction in a breast cancer cell line in response to hypoxia resulted in an interaction with the stem cell regulatory gene Notch-3 [23]. Furthermore, abnormal p66Shc expression has been found in several cancers including breast [24], prostate [25], esophageal [26], and colon [27]. In a recent study, p66Shc was simultaneously found to promote immune suppression through the activation of STAT3 and impair immune surveillance by preventing activation of STAT1 in breast cancer cells [28]. Furthermore, in chronic lymphocytic leukemia (CLL) B cells a positive feedback loop was identified which coupled p66Shc and STAT4 expression whereby STAT4 regulated p66Shc expression by interacting with specific binding sites in the p66Shc promoter [10]. In a study of myocardial injury in mice, p66Shc was identified as a regulator of myocardial injury through its interaction with STAT3 [29]. STAT3 is a redox sensitive transcription factor that has been found to function as a switch between inflammation and cancer [30,31]. Activation of STAT3 through its phosphorylation to p-STAT3 is thought to be involved in its role in the development and progression of tumors [5]. Sustained levels of p-STAT3 have been found in gastric cancer [32], pancreatic cancer [33], prostate cancer [34], breast cancer [35] and other malignant cancers [36,37]. In this study we investigated the clinical significance of p66Shc levels in HCC and endeavored to discover possible roles for p66Shc in the development of HCC potentially through activation of the STAT3 pathway.

2.3. Cell culture and generation of conditioned medium The human HCC cell lines (Huh7, HepG2, and SK-HEP1) and the normal liver cell line LO2 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. Human acute monocytic leukemia THP-1 cells were obtained from the China Center for Type Culture Collection, Wuhan, China. HepG2, LO2, and THP-1 cells were cultured in RPMI 1640 medium (Gibco, Grand land, NY, USA) containing 10% fetal bovine serum (Gibco). Huh7 cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum. SK-HEP1 cells were cultured in MEM medium (Gibco) with 10% fetal bovine serum. All of the cell lines were maintained in medium supplemented with 1% penicillin/streptomycin (Gibco) at 37 °C, in a humidified atmosphere of 5% CO2. Conditioned medium (CM) was obtained from the treatment of THP1 cells with phorbol-12-myristate 13-acetate (PMA) to produce activated macrophages. Briefly, THP-1 cells were stimulated with 50 nM PMA (Sigma-Aldrich for 24 h. The PMA-containing medium was removed after 24 h and the cells were washed three times in PBS to thoroughly remove the PMA. The cells were then kept in complete culture media for a further 24 h after which time the CM was collected. After collection the CM was filtered through a 13-mm syringe filter (0.45 μm pore size, Thermo Scientific, Waltham, MA, USA) and mixed with an equal volume of fresh MEM medium and added to the cell culture to be studied.

2. Materials and methods 2.1. Patient characteristics and tissue specimens Patients were recruited from Zhongshan Hospital, Fudan University, Shanghai, China. The tissue microarray (TMA) cohort was comprised of 312 patients with completed survival data who underwent hepatectomy between November 2005 and December 2012. Tumor differentiation was defined according to the Edmondson grading system [38]. The qRT-PCR cohort consisted of 15 surgical cases obtained between December 2008 and April 2011. Informed consent was obtained from all patients and the study was approved by, and conformed to all criteria set, by the local Ethics Committee.

2.4. RNA extraction and qRT-PCR analysis Total RNA was extracted using the Trizol reagent as per the manufacturer (Invitrogen). cDNA was prepared using the HiScript Reverse Transcriptase (RNase H) (VAZYME, Nanjing, China). Quantitative PCR (qPCR) was performed using the QuantStudio 6 System (Applied Biosystems, Foster City, CA, USA) with SYBR Green Master Mix (VAZYME). The following primers were used for qRT-PCR. p66Shc; forward, 5'-AAGTACAATCCACTCCGGAATGA-3'; reverse, 5'-GGGCCCC AGGGATGAAG-3′. CXCL1; forward, 5'-GGGAATTCACCCCAAGAAC ATC-3′; reverse, 5'-GGATGCAGGATTGAGGCAAGC-3′, CXCL2, forward, 5'-GTGTGAAGGTGAAGTCCCCC-3′; reverse, 5'-TCTTAACCATGGGCGA TGCG-3′, CXCL5, forward, 5'-CAGACCACGCAAGGAGTTCA-3′; reverse,

2.2. Tissue microarray construction, immunohistochemistry, and immunofluorescence The HCC TMAs were designed by a contract service at the Shanghai Outdo Biotech Co., LTD, Shanghai, China. Paraffin-embedded tissues were used for immunohistochemistry (IHC) and immunofluorescence (IF). Serial tissue sections were firstly deparaffinized and then rehydrated. The sections were then subjected to heat-induced epitope retrieval in a microwave oven. Treatment of the sections with 3% hydrogen peroxide was performed to quench endogenous peroxidase activity. Non-specific binding was blocked by incubation with 5% FBS 2

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2.9. In vitro invasion and migration assay

5'-CCGTTCTTCAGGGAGGCTAC-3′. GADPH was used as an endogenous control in the qRT-PCR assay and was amplified with the following primers; -3';, GAPDH, forward, 5'-TCAAGAAGGTGGTGAAGCAGG-3'; reverse, 5'-TCAAAGGTGGAGGAGTGGGT-3', relative fold changes were calculated using the 2−ΔΔCt method.

The invasive and migratory potential of the cells was assayed using transwell chambers (8 μm pore; BD Biosciences). For the invasion assay, 1 × 105 cells suspended in 100 μl of serum free medium were added to the upper chamber of the inserts, which were coated with Matrigel (BD Biosciences). Chemoattractant (500 μl of fetal bovine serum) was added to the lower chamber. The wells were then incubated for 24 h at 37 °C in 5% CO2. Subsequently, non-invading cells on the upper surface were wiped off with a cotton swab and cells that had invaded the lower side of the membrane were fixed with methanol, stained with 0.1% crystal violet, air dried, and then photographed. For the migration assay, 5 × 104 cells suspended in 100 μl serumfree medium were placed in the upper chamber of each insert without Matrigel, and 500 μl of fetal bovine serum was added to the lower chamber. After 16 h incubation at 37 °C in 5% CO2, the remaining cells in the upper chamber were wiped away with a cotton swab. Cells that had migrated to the lower chamber were fixed with methanol, stained with 0.1% crystal violet, and air dried. The experiments were performed in triplicate, and three fields were counted per filter in each group.

2.5. Western blotting Cells or tissues were lysed in RIPA lysis buffer (Beyotime, Jiangsu, China) and the protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, MA, USA). 30 μg of lysate proteins were separated on a 12% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by immersing the membranes in 5% non-fat milk in PBS for 1 h at room temperature. The membranes were then incubated with primary antibodies for Shc1 (1:2000) (Abcam, Cambridge, UK), STAT3 (1:1000), Phospho-Stat3 (Ser727) (1:1000), Phospho-Stat3 (Tyr705) (1:1000) (Cell Signaling Technology, MA, USA), GAPDH (1:1000) (Santa Cruz, CA, USA). After washing, the blots were incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using an enhanced chemiluminescence Western Blot analysis system (Pierce).

2.10. Mouse xenograft model 2.6. Plasmid construction and p66SHC knockdown

In order to generate subcutaneous tumors, SK-HEP1 cells infected with either Scramble, sh-p66shc #1, or sh-p66shc #2 (1 × 106 cells/ mouse) were injected subcutaneously into the flanks of 4–5 week old female athymic BALB/c nude mice (Shanghai Slac Laboratory Animal Co., China). All of the mice were housed under air-filtered pathogenfree conditions. Growth of tumors was monitored with digital calipers every 5 days. Tumor volumes were estimated using the formula: volume (mm3) = (width)2 × length/2 and tumor growth was plotted against time. After 35 days, the animals were sacrificed and the tumors were dissected. Tumor tissues were fixed with 10% formalin and embedded in paraffin. Sections (4 μm) were prepared and stained with hematoxylin and eosin. Tumor sections were obtained and stained with specific antibodies against Ki-67 in order to detect cell proliferation (1:500, Abcam). Animal studies were approved by the Institutional Animal Care and Use Committee of the Fudan University, Shanghai, China.

To stably knockdown p66Shc, two shRNA sequences were designed and were as follows: 5'-UGAGUCUCUGUCAUCGCUG-3′ (sh-p66shc #1) and 5'-CGAUAGUCCCACUACCCUG-3′ (sh-p66shc #2). In addition, lentivirus shRNA vector of scrambled oligomer (5'-UUCUUCGAACGU GUCACGUTT-3') (Scramble) was used as a negative control. The sequences were subsequently cloned into the lentivirus vector pGCSILGFP (Genechem, Shanghai, China) and verified with DNA sequencing. The resulting lentivirus vectors together with pHelper1 and pHelper2 vectors were co-transfected into 293T cells for 48 h using Lipofectamine 2000 (Invitrogen). After incubation for 48 h, the cultured lentiviral supernatant was collected and purified. After the titers were determined, the resultant lentivirus vectors containing Scramble, shp66Shc #1, or sh-p66Shc #2 were used to infect SK-HEP1 cells. Stable cell lines with knockdown of p66Shc were established by selection with 3 μg/ml puromycin hydrochloride (Sigma-Aldrich) for four weeks.

2.11. Statistical analysis

2.7. MTT assay

The Student's t-test was used to statistically analyze the differences in mean values between two groups. One-way ANOVA was used to determine if the differences between mean values in multiple groups were statically significant. All data shown are expressed as the mean ± SD. SPSS version 17.0 and GraphPad Prism 5.0 software were used for the statistical analysis. Shc1 expression was categorized as high or low using the median Shc1 H-score, and the Chi-square were used to determine the association of Shc1 expression with clinical and pathological variables. The Kaplan-Meier method was used to calculate survival curves that were compared by a log-rank test. Results were considered statistically significant when p < 0.05 and all p values are two-tailed.

Cells were seeded onto 96-well plates at a concentration of 1 × 104 cells/well in 100 μl of fresh culture medium. After overnight incubation, the cells were then further incubated for 24 h either with 100 μl of CM from PMA-activated THP-1 cells (CM+) or in 100 μl culture medium (CM–). Subsequently, 20 μl of MTT (0.5 mg/ml, Sigma) was added to each well and the plates were incubated for an additional 4 h. Culture medium from all plates was then removed and 150 μl of DMSO reagent was added to dissolve precipitates. Lastly, the optical density (OD) of each well was measured with a microplate spectrophotometer (ELx800, Bio-TEK, Winooski, VT, USA) at a wavelength of 570 nm. All experiments were completed in triplicate.

3. Results 2.8. Colony formation assay 3.1. Increased expression of Shc1 in human HCC tissues predicts a poor prognosis

Cells were cultured with or without CM for 24 h and were subsequently trypsinized and counted. These cells were then seeded onto 35mm culture dishes (1000 cells/well) and cultured in a complete medium for 14 days at 37 °C in 5% CO2. After the incubation period the medium was removed and the plates washed in PBS and fixed in 4% paraformaldehyde. After again washing in PBS, cells were stained with crystal violet and photographed. The experiments were performed in triplicate.

In order to determine if there was a correlation between Shc 1 expression levels and clinicopathological factors in HCC, we constructed a tissue microarray containing 312 pairs of HCC and adjacent non-tumor tissues for IHC and IF staining. Shc1 staining was enriched in the cytoplasm (Fig. 1A), which was confirmed by IF staining (Fig. 1C), and Shc1 staining was significantly greater in the HCC tissues (p < 0.05) 3

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Fig. 1. Upregulation of p66Shc in HCC tissues. (A) Representative immunohistochemistry (IHC) images of p66Shc IHC staining in HCC and peritumoral tissues in tissue microarray. (B) Relative IHC staining of p66Shc in paired HCC and peritumoral tissue samples are shown. Statistical significance was determined by student's ttests. (C) IF staining for Shc1 in HCC tissues was performed. The nuclei are counterstained with DAPI (blue). (D) The overall survival and disease-free rates of patients of HCC were compared between the low-Shc1 and high-Shc1 groups. (E) Relative expression of p66Shc mRNA in 15 paired HCC and peritumoral tissues were evaluated by qRT-PCR. (F) Expression of p66Shc protein in HCC cancer (T) and peritumoral tissues (P) tissues from four HCC patients, determined by Western blot assay.

4

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cells (p < 0.01). Western blotting confirmed that p66Shc protein levels were greater in the HCC cell lines (Fig. 2B) with SK-HEP1 cells exhibiting the densest band. To investigate the effects of p66Shc on cell growth and motility in HCC cells, we stably silenced p66Shc expression in SK-HEP1 cells using lentivirus transfection. Western blotting showed that both sh-p66shc #1 and sh-p66shc#2 significantly inhibited p66Shc protein expression in SK-HEP1 cells (Fig. 2C). The MTT assay showed that knockdown of p66Shc suppressed the proliferation of SK-HEP1 cells when compared with control cells (Fig. 2D). Similarly, the colony formation assay results showed that colony formation capacity was dramatically reduced in p66Shc knockdown cells (p < 0.05) (Fig. 2E). Based on the data obtained above the role of p66Shc knockdown in cell migration and invasion was assessed. The results of these assays (Fig. 2E) showed that compared with control SK-HEP1 cells, migration and invasion ability was significantly repressed in SK-HEP1 cells with knockdown of p66Shc. The results presented here show that knockdown of p66Shc inhibits cell growth and motility of HCC cells in vitro.

Table 1 Correlation between Shc1 expression levels and clinicopathological factors in HCC (n = 312). Variable

Low-Shc1 (115)

High-Shc1 (197)

Male Female

91 24

179 18

< 50 ≥50

44 71

61 136

<5 ≥5

25 90

25 172

Solitary Multiple (≥2)

101 14

156 41

Well Moderate Poor

4 111 0

4 191 2

Positive Negative

92 23

160 37

Present Absent

69 46

122 75

p Value 0.003**

Gender

Age (years)

0.188

0.036*

Size (cm)

Tumor nodes

0.053

Differentiation

0.414

HBsAg

0.792

Cirrhosis

3.3. p66Shc knockdown attenuates cell growth and motility of HCC cells exposed to the conditioned media from PMA-activated THP-1 cells

0.736

It is increasingly evident from numerous studies that the inflammatory TME facilitates tumor progress, and macrophages play an important role in the inflammatory milieu of the TME [39]. Furthermore, it has been proven that CM from PMA-activated THP-1 cultures enhances the vitality of HCC cells [40]. Therefore, we investigated whether the CM from PMA-activated THP-1 cells had an impact on the cell growth and motility of HCC cells. SK-HEP1 cells were cultured with or without CM from PMA-activated THP-1 cells for 24 h. The MTT assay showed that SK-HEP1 cells incubated with CM exhibited significantly increased proliferative ability compared to the cells grown without CM (p < 0.05) (Fig. 3A). Knockdown of p66Shc inhibited the proliferative ability of SK-HEP1 cells in the presence of CM. The colony formation assays revealed that culture in the presence of CM resulted in increased colony formation capacity in the SK-HEP1 cells (Fig. 3B). Furthermore, the number of colonies formed by both of the p66Shc knockdown transfected cells was significantly less than those formed by the Scramble cells cultured in CM (p < 0.05) (Fig. 3 B). The migration and invasion assays were performed to evaluate motility in cells cultured with and without CM (Fig. 3C). For the migration and invasion assay SK-HEP1 cells grown in CM showed significantly increased migration and invasion capacity when compared with SK-HEP1 cells grown without CM (p < 0.01). In the p66Shc knockdown cell cultures grown with CM there was a significant decrease in cell migration and invasion when compared with the cells transfected with Scramble (p < 0.01). The results presented here show that knockdown of p66Shc reduces cell growth and cell motility in response to CM from activated macrophages.

0.015*

Recurrence Yes No

56 59

123 73

Yes No

86 29

120 77

0.013*

Death

Low-Shc1, H-score≤89.23; ** p < 0.01, Chi-square test.

High-Shc1,

H-score > 89.23;

*

p < 0.05,

(Fig. 1B). As shown in Table 1, of the 312 patients studied 115 exhibited low-Shc 1 (H-score ≤ 89.23) while 197 exhibited high-Shc1 (Hscore > 89.23). Of the 197 patients with high-Shc1 179 (> 90%) were male which was statistically significant (p = 0.003). A larger tumor size of ≥5 cm was associated with a high-Shc 1 level and was statistically significant (p = 0.036). Intriguingly, the presence of high-Shc 1 was associated with tumor recurrence and death (p = 0.015 and 0.013 respectively). However, there was no statistical correlation between Shc1 levels and other clinicopathologic parameters, such as age, number of tumors, differentiation, and Cirrhosis. The overall survival and diseasefree rates of patients with HCC were compared between the low-Shc1 and high-Shc1 groups and are shown in Fig. 1D. These results show that overall survival and disease-free survival were significantly decreased in the high-Shc1 group. To determine the level of p66Shc in HCC, the relative expression of p66Shc mRNA in 15 paired HCC and peritumoral tissues was evaluated by qRT-PCR. As shown in Fig. 1E, the expression of p66Shc mRNA was significantly greater in the tumor tissues when compared with the matched peritumoral controls (p < 0.01). Western blot analysis further confirmed p66Shc expression was increased in HCC tissues at the protein level (Fig. 1F). All three isoforms of the Shc1 protein, p46, p52, and p66, were upregulated in HCC tissues. Taken together these results (Table 1 and Fig. 1) show that high-Shc 1 levels in HCC patients are associated with male patients with HCC and predict a larger tumor size associated with reduced overall survival and predict a poor prognosis.

3.4. p66Shc promotes activation of STAT3 signaling SK-HEP1 cells infected with lentivirus carrying Scramble or either of the sh-p66Shc knockdown sequences (sh-p66shc #1or sh-p66shc#2) were cultured with or without CM from PMA-activated THP-1 cells for 24 h. To determine if p66Shc knockdown had an effect on STAT3 activation, the phosphorylation state of STAT3 was analyzed by Western blot. In Fig. 4A it can be seen that the protein levels of p66Shc and total STAT3 was unchanged when cells were cultured with CM. Both the relative levels of p-STAT3 (Y705) and p-STAT3 (S727) are increased in the presence of CM. In addition, p-STAT3 (S727) was reduced in the p66Shc silenced cells both in the absence and presence of CM when compared with the control and Scramble treated cells. However, levels of p-STAT3 (Y705) were unaffected by p66Shc knockdown both in the absence and presence of CM (Fig. 4A). These results indicate that knockdown of p66Shc does not result in a difference in the levels of

3.2. p66Shc knockdown inhibits cell growth and motility in HCC cells We detected mRNA and protein levels of p66Shc mRNA expression in the HCC cell lines (Huh7, HepG2, and SK-HEP1) and in the immortalized human liver cell line LO2. As depicted in Fig. 2A, p66Shc mRNA expression was significantly greater in the HCC cell lines (p < 0.05) when compared with the LO2 cells. Moreover, of the three HCC cell types p66Shc mRNA levels were the highest in the SK-HEP1 5

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Fig. 2. Inhibition of p66Shc suppresses HCC cell growth and motility. (A and B) Relative expression of p66Shc mRNA (A) and protein (B) were determined by qRTPCR analysis and Western blot in human HCC cell lines. (C) Western blot analysis of p66Shc in SK-HEP1 cells infected with lentivirus carrying either Scramble or shp66Shc knockdown sequences (sh-p66shc #1 or sh-p66shc #2). (D) Cell proliferation was evaluated using the MTT assay. (E) Colony formation assays were performed to evaluate cell growth in SK-HEP1 cells infected with lentivirus carrying either Scramble or sh-p66Shc knockdown sequences (sh-p66shc #1 or sh-p66shc #2). (F) Transwell migration and invasion assays were performed to evaluate the motility of SK-HEP1 cells infected with lentivirus carrying either Scramble or shp66Shc knockdown sequences (sh-p66shc #1 or sh-p66shc #2).

transfected with Scramble when cultured in the absence of CM (Fig. 4D). Furthermore, culture of the cells with CM, although it did produce increased CXCL5 expression compared to cells not cultured in CM (p < 0.01), was not dependent on the presence or absence of functioning p66Shc as there was no significant difference between the CXCL5 mRNA levels between Scramble, or either of the 2 p66Shc knockdown cell cultures. These results would appear to indicate that CXCL5 upregulation is not dependent on the presence of functioning p66Shc.

total STAT3 but does result in a decrease in the level of p-STAT3 (S727), and that functioning p66Shc is required for p-STAT3 (S727) activation in response to an inflammation-like environment. In order to determine the effect of p66Shc on the levels of cytokines, the relative mRNA expression of CXCL1, CXCL2, and CXCL5 was determined by qRT-PCR. As can be seen from the results presented in Fig. 4B and C, in the absence of CM, the relative expression of CXCL1 and CXCL2 mRNA was significantly lower in cells transfected with p66Shc knockdown lentiviruses when compared with cells transfected with Scramble (p < 0.05). When the cells were cultured in the presence of CM there was a substantial increase in CXCL1 and CXCL2 mRNA expression in the control and Scramble treated cells (p < 0.01). Similarly, the expression of CXCL1 and CXCL2 mRNA was also significantly decreased in cells transfected with p66Shc knockdown lentiviruses when compared with cells transfected with Scramble (p < 0.01). These results indicate that p66Shc is required for the upregulation of CXCL1 and CXCL2 in response to an inflammation-like environment. When the relative mRNA expression levels of CXCL5 were examined no significant difference in expression was found between the cells transfected with p66Shc knockdown lentiviruses compared with cells

3.5. p66Shc knockdown suppresses cell growth and STAT3 activation in vivo To determine if p66Shc knockdown suppresses tumor growth in vivo we subcutaneously injected nude mice with SK-HEP1 cells infected with lentivirus carrying either Scramble or sh-p66Shc knockdown sequences (sh-p66shc #1 or sh-p66shc#2). The growth of tumors was then assessed for 35 days post-injection. As shown in Fig. 5A and B the knockdown of p66Shc reduced the tumor volume significantly. In addition, the weight of the tumors was also significantly reduced in mice with sh-p66Shc knockdown when compared with that of scramble cells 6

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Fig. 3. Inhibition of p66Shc attenuates cell growth and motility of CM-stimulated HCC cells. SK-HEP1 cells infected with lentivirus carrying either Scramble or either of the two shRNA knockdown sequences (sh-p66shc #1 or sh-p66shc#2) were cultured with or without conditioned media (CM) from PMA-activated THP-1 cells for 24 h. (A) The MTT assay was performed to detect cell proliferation. (B) Colony formation assays were performed to evaluate cell growth. (C) Transwell migration and invasion assays were performed to evaluate cell motility.

Fig. 4. P66Shc promotes activation of STAT3 signaling. SK-HEP1 cells infected with lentivirus carrying either Scramble or either of the two shRNA knockdown sequences (sh-p66shc #1 or sh-p66shc#2) were cultured with or without conditioned media (CM) from PMA-activated THP-1 cells for 24 h. (A) The total STAT3 and phosphorylation of both Y705 and S727 STAT3 sites in tumor tissues was analyzed by Western blot analysis. (B–D) The relative expression of CXCL1 (B), CXCL2 (C), and CXCL5 (D) mRNA was obtained using qRT-PCR. 7

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Fig. 5. Inhibition of p66Shc suppresses cell growth and motility in vivo. A mouse xenograft tumor model was used. Mice received subcutaneous injections of 1 × 107 SK-HEP1 cells infected with lentivirus carrying either Scramble or either of the two shRNA knockdown sequences (sh-p66shc #1 or sh-p66shc#2). (A) The tumor growth curve during the 35 day study period is shown. Data are shown as mean ± SEM. (B) Harvested mouse xenograft tumors were evaluated 35 days postinjection and representative images are shown. (C) Tumor weight was evaluated 35 days post-injection. (D) The representative images of H&E stain and IHC of Ki67 of tumor tissues are shown. (E) Expression of p66Shc and total STAT3, and phosphorylation of both Y705 and S727 STAT3 sites in tumor tissues was analyzed by Western blot analysis.

of STAT3 which in turn is an important contributor to tumorigenesis [42]. According to the clinical data, this study shows that p66Shc expression was higher in human HCC tissues than adjacent non-tumor tissues. HCC patients with high p66Shc levels had a worse prognosis and higher recurrence rates than those with low p66Shc levels. Furthermore, patients with high p66Shc levels were more likely to be male and have larger tumors. These clinical results revealed that determination of p66shc levels could serve as a prognostic indicator for HCC and is the first time that a correlation between p66Shc and HCC has been reported. In our initial investigations we found that levels of the three Shc isoforms increased in HCC tissue. Although, the three isoforms of Shc are derived from the same gene, p66shc possesses an extension of 110 amino acids at its N-terminus. The addition of the extension has conferred a distinct role on p66Shc that is not shared by the two shorter isoforms (p46Shc and p52Shc) [43]. There is evidence that both p46Shc and p52Shc are associated with HCC. Previously, Yukimasa et al. showed that p46Shc expression and its phosphorylation may be strongly indicative of malignant transformation, tumor invasion, and metastasis in HCC [44]. In addition, Zhang et al. recently showed that the p52Shc/ERK1/2 pathway mediated malignant behavior of HCC cells [45]. In the basic studies performed we found that knockdown of p66Shc could suppress HCC cell growth and motility in vitro. In addition, we found that knockdown of p66Shc expression inhibited tumor growth in vivo. Furthermore, when HCC cells were exposed to CM, from activated macrophages in a rudimentary simulation of the TME, it was found that knockdown of p66Shc reduced the CM-induced growth and motility of HCC cells. In experiments investigating the underlying mechanism of action of p66Shc, it was found that knock down of p66Shc reduced the activation of STAT3 in vitro and in vivo. In addition, the knockdown of p66Shc resulted in a reduction in the phosphorylation of STAT3 on serine 727

(p < 0.05) (Fig. 5C). In order to examine cell proliferation in the tumor tissues IHC staining of Ki67 was performed. As can be seen in Fig. 5D knockdown of p66Shc in these mouse tumors resulted in decreased cell proliferation as visualized by Ki67 staining. Finally, expression of p66Shc and the phosphorylation status of STAT3 in mouse tumor tissues were analyzed by Western blot. As shown in Fig. 5E, in tumors with Scramble or p66Shc knockdown there was no difference in the protein levels of total STAT3 and p-STAT3 (Y705). However, when the relative levels of p-STAT3 (S727) were compared, it can be seen that knockdown of p66Shc results in a reduced presence of p-STAT3 (S727). These results provide in vivo evidence that p66Shc was capable promoting of tumor growth in vivo and acted as an oncogene in HCC, indicating that p66Shc is a potential target for HCC molecular therapy. 4. Discussion Previous studies have found that abnormal expression of p66Shc was associated with several cancers including breast [24], prostate [25], esophageal [26], and colon [27]. However, the implications of abnormal p66Shc expression for tumorigenesis are yet to be fully determined. One study found that p66Shc promoted androgen-independent cell growth through generation of reactive oxygen species (ROS) [41]. Furthermore, the finding that the induction of p66Shc in a breast cancer cell line in response to a hypoxic environment and the subsequent interplay between p66Shc and the stem cell regulatory gene Notch-3 could shed some light on how cancer cells survive and proliferate in the hypoxic environment which is typical of the TME [23]. Although the evidence for an involvement of p66Shc in tumorigenesis is increasing the molecular pathway to which it contributes in this process remains elusive. In this study we report, the clinical significance of p66Shc in HCC and its correlation with cell growth, motility, and prognosis. We also reveal a significant role for p66Shc in the activation 8

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order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We further confirm that any aspect of the work covered in this manuscript that has involved either experimental animals or human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected].

(S727) in vitro and in vivo. Previous studies have found that STAT3 is activated by the phosphorylation of tyrosine 705 (p-STAT3 [Y705]), which then forms a dimer, translocates to the nucleus, binds to the promoters of genes involved in cell proliferation, cell survival, angiogenesis, and metastasis and induces their expression thus contributing to tumorigenesis [46]. However, phosphorylation of another domain, the serine domain (S727), is postulated to be essential for maximal STAT3 transcriptional activity [47]. The phosphorylation of S727 is thought to function as an additional layer of regulation of STAT3mediated gene expression [47]. Furthermore, there is evidence to suggest that phosphorylation of S727 before Y705 negates the requirement for phosphorylation of Y705 [47]. The finding in our study that p66Shc activates p-STAT3 (S727) preferentially to Y705 suggests a mechanism whereby p66Shc contributes to tumorigenesis through the sustained activation of STAT3 at S727. In agreement with our results, a recent study found that p66Shc deletion was found to reduce the phosphorylation of STAT3 at S727 but had no effect on phosphorylation at Y705 [29]. In addition, it was found that phosphorylation of STAT3 at Y705 drives p66Shc to the nucleus whereas phosphorylation at S727 drives p66Shc to the mitochondria where it prevents mitochondrial swelling and apoptosis [29]. Also, STAT3 has been recognized as an important regulator of mitochondrial metabolism independent of its function as a transcription factor [47]. Our results provide additional evidence that the interaction between p66shc and STAT3 is through the differential phosphorylation of the Y705 and S727 sites. Phosphorylated STAT3 has been recognized as a critical regulator of tumor-associated inflammation because, in addition to the promotion of tumor cell proliferation, survival, angiogenesis, and metastasis, activated STAT3 can negatively regulate immunogenic cells in the TME and thus restrain anti-tumor immune responses [8]. In this study we performed preliminary experiments to determine if p66Shc knockdown resulted in a change in HCC CXCL1, CXCL2, and CXCL5 expression. Our results showed that although CXCL1 and CXCL2 expression levels were reduced by knockdown of p66Shc CXCL5 levels were not affected. CXCL1 and CXCL2 are both cytokines released in the early stages of inflammation which recruit neutrophils to the inflammation site [48]. In this way it would appear that p66Shc indirectly activates their expression and thus promotes the inflammatory TME. Our results showed that the expression of CXCL5 was unaffected by the knockdown of p66Shc and this is in agreement with another study that showed that p66Shc deficiency was not associated with inflammation directly as it did not exert its effects through the activation of CXCL5 [47]. However, STAT3 and the transcription factor Sp1 have been found to form a complex which enhances the inflammatory response [47]. Increased phosphorylation of S727 recruits Sp1 in endothelial cells treated with IL-6 cytokine stimulation [47]. Furthermore, it has been shown that CXCL1 and CXCL2 both contain Sp1 promoter binding sites but CXCL5 does not [49] and this may explain why in our study we did not find an increase in CXCL5 expression. In conclusion, we have provided novel insight into the association of p66Shc expression in malignant behavior, recurrence, and prognosis in HCC patients. Our findings revealed that p66Shc is upregulated in HCC tissues, and could play a significant role in tumor progression in HCC through activation of the STAT3 pathway by the phosphorylation of STAT3 at S727. Thus, these findings together have defined p66Shc as an oncogene in HCC, which may be a potential prognostic biomarker and therapeutic target for HCC patients.

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Conflict of interest form We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the 9

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