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RESEARCH COMMUNICATION
Department of Pharmacology and Cancer Biology, Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710, USA
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Abstract |
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[Keywords: Ras; oncogene; IL6; cytokine; cancer]
Received March 6, 2007; revised version accepted June 8, 2007.
). On the other hand, secreted proteins are druggable, typically with neutralizing antibodies (Adams and Weiner 2005). We therefore speculated that inhibiting secreted proteins induced by Ras might block Ras oncogenesis.
The cancer most associated with oncogenic Ras mutations is pancreatic (Downward 2003). Proteins elevated in the serum of pancreatic cancer patients may correspondingly be the product of oncogenic Ras activity. One such protein elevated in pancreatic cancer patients is IL6 (Barber et al. 1999; Wigmore et al. 2002; Ebrahimi et al. 2004). IL6 is a pleiotropic cytokine functioning in inflammation, immunity, bone metabolism, neural development, reproduction, and hematopoiesis (Keller et al. 1996), but it has also been implicated in the etiology of some cancers (Trikha et al. 2003). Given the alluring link of elevated IL6 levels in patients with a cancer characterized by Ras mutations and the observation that Ras-induced secretion of another cytokine, IL8, is important for tumor growth of HeLa cells overexpressing oncogenic Ras (Sparmann and Bar-Sagi 2004), we examined whether IL6 plays any role in Ras-mediated cancers.
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Results and Discussion |
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Normal human kidney cells genetically transformed with SV40 T/t-Ags, the telomerase catalytic subunit hTERT, and a 4-hydroxytamoxifen (4-OHT) inducible ER:RasG12V fusion protein were assayed for secreted IL6 levels in the absence of 4-OHT, and hence, no ER:RasG12V protein, and presence of 4-OHT, and hence, the presence of ER:RasG12V, which converts these cells to a tumorigenic state (Lim and Counter 2005). Upon induction of ER:RasG12V by 4-OHT (Fig. 1A, top panel), the level of secreted IL6 was elevated almost 40-fold (Fig. 1A, bottom panel). Thus, induction of a tumorigenic state by oncogenic Ras is associated with an increase in IL6 secretion.
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). To address whether induction of IL6 secretion occurred in other cell types, IL6 secretion was compared with the Ras status and tumorigenic phenotype of four different cell types that retained a constant genetic background (O’Hayer and Counter 2006). Both mRNA and protein levels of IL6 were greatly increased in normal primary human kidney cells, fibroblasts, myoblasts, and mammary epithelial cells expressing T/t-Ag and hTERT that were driven to be tumorigenic by ectopic expression of RasG12V compared with the nontumorigenic cells lacking RasG12V (Fig. 1B). Thus, an oncogenic Ras-mediated tumorigenic state is associated with elevated levels of IL6 in multiple cell types.
Ras-induced secretion of IL6 is required for human tumor cell growth in vivo
To test whether the Ras-induced secretion of IL6 is required for Ras oncogenic function, IL6 levels were stably reduced >90% by IL6 short hairpin RNA (shRNA) in the tumorigenic human kidney cells expressing T/t-Ag, hTERT, and RasG12V (Fig. 1C). The resultant cells or the scramble control counterpart were then tested for tumor growth in immunocompromised mice. While scramble control cells rapidly formed tumors, reaching maximum tumor volume within 
20 d, IL6 shRNA-treated cells barely generated palpable masses in this time span (P < 0.001) (Fig. 1D,E). Even though tumors eventually did arise (data not shown), IL6 was re-expressed in these tumors (Fig. 1F), strongly suggesting that a loss of IL6 must be overcome for tumor growth.
We validated these results with two more independent IL6 shRNA sequences. Identical to the first IL6 shRNA, knockdown of IL6 by these two other sequences (Fig. 1C) blocked tumor growth of the aforementioned RasG12V-transformed human kidney cells (Fig. 1E). To address whether IL6 is required for Ras-driven growth independent of cell type, IL6 was knocked down by shRNA (Supplementary Fig. 1A,D) in the aforementioned human fibroblasts and myoblasts engineered to be tumorigenic by the expression of T/t-Ags, hTERT, and RasG12V (O’Hayer and Counter 2006) and again found to reduce tumor size by at least 97% compared with scramble control cells (Supplementary Fig. 1B,C,E,F). Knockdown of IL6 thus presents a formidable barrier to Ras-induced human tumor growth of cells derived from different lineages.
IL6–/– mice are highly resistant to spontaneous induced Ras-driven tumors
To address whether IL6 is also required for Ras-mediated tumor growth in a system that recapitulates the spontaneous process of tumorigenesis, we tested whether IL6–/– knockout mice are resistant to carcinogen-induced tumors. Premalignant papillomas with a high level of Ras mutations are induced by topical application of the carcinogen 7,12-Dimethylbenzanthracene (DMBA), followed by repetitive application of 12-O-tetradecanoylphorbol-13-acetate (TPA) (Quintanilla et al. 1986). Thus, IL6+/+ and IL6–/– mice were treated topically with DMBA and then TPA for 20 wk, and tumor growth monitored. Tumors appeared within 12 wk of initiation in IL6+/+ mice, and by termination of the experiment, 93% of the mice had tumors, with an average of 2.4 tumors per mouse (Fig. 2A–C). In sharp contrast, tumors appeared 2 wk later in the IL6–/– mice (Fig. 2A,B), and by the termination of the experiment, only 27% of the mice had tumors (Fig. 2B) and the average number of tumors dropped to 0.5 per mouse (P < 0.01) (Fig. 2C). Perhaps most telling, the total tumor volume per mouse at week 20, a measurement of both the size and number of tumors dropped almost 30-fold in IL6–/– mice (P < 0.05) (Fig. 2D). In agreement, a 1-wk delay in tumor onset and a 20% reduction in mice with tumors had been described for DMBA/TPA-treated IL6–/– mice, although in this case, a more aggressive carcinogen protocol was used (Suganuma et al. 2002). Thus, loss of IL6 inhibits spontaneous formation of Ras-driven tumors.
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To address how Ras-induced secretion of IL6 promotes tumor growth in vivo, we tested whether the tumor cells were themselves the target of IL6. However, we did not find any evidence for autocrine signaling. RasG12V-transformed human kidney cells, which require IL6 for tumor growth, do not express detectable IL6 receptor (IL6R) (Fig. 3A), and knockdown of IL6 in these cells did not, when compared with vector control cells, have any effect on cell proliferation under normal serum (Fig. 3B, top panel) or in the stress condition of low serum (Fig. 3B, middle panel) or growth in soft agar (Fig. 3C), nor did addition of exogenous IL6 foster the growth of the same cells in the absence of Ras (Fig. 3B, bottom panel). We thus explored the possibility that Ras-induced secretion of IL6 acts in a paracrine fashion to promote Ras tumorigenesis. Immunohistological staining revealed that tumors eventually arising from IL6 shRNA-treated RasG12V-transformed human kidney cells did not exhibit any gross histological differences, or changes in cell proliferation, as assessed by Ki67 and phospho-histone H3 staining, or apoptosis, as detected by the TUNEL assay, compared with scramble control tumors (P > 0.05) (Fig. 3D). This could mean that either loss of IL6 has no effect on apoptosis or proliferation, or that by the time we are able to study these tumors, the observed reactivation of IL6 (Fig. 1F) masked these effects. Nevertheless, we did find that CD31-positive (endothelial) cells were reduced almost 18-fold in IL6 shRNA-treated tumors (P < 0.01) (Fig. 3D). In agreement, IL6 has been shown to promote angiogenesis (Cohen et al. 1996; Wei et al. 2003; Huang et al. 2004; Loeffler et al. 2005; Nilsson et al. 2005). However, endothelial cells typically do not express detectable levels of IL6R (Supplementary Fig. 2) (Romano et al. 1997) or respond to IL6 (Podor et al. 1989; Sironi et al. 1989), arguing that the reduction of CD31-positive cells by knocking down IL6 in tumorigenic cells is indirect. This paracine effect must also be local, as tumors arising from scramble control cells on one flank of the mice failed to promote tumorigenic growth of IL6 shRNA-treated cells on the opposite flank (Fig. 1D).
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To expand our studies to more clinically relevant settings, we tested whether stable knockdown of IL6 (Fig. 4A, top panels) inhibited the tumorigenic growth in the human pancreatic cancer cell lines CFPac-1, Capan-1, and HPAC that contain an oncogenic K-Ras allele (Kita et al. 1999; Moore et al. 2001; Lim et al. 2006), as such cell lines better model the genetic chaos of human cancers that is thought to underlie the variability in the effectiveness of cancer therapies. While knockdown of IL6 had no obvious effect on CFPac-1 cells, it did cause necrosis in the tumors arising from Capan-1 cells and retarded the tumor growth of HPAC cells compared with scramble control-treated cells (Fig. 4A, bottom panels). Thus, IL6 is important for tumorigenic growth in two of the three tested human mutant K-Ras-positive cancer cell lines, suggesting that an anti-IL6 therapy could be effective in the heterogeneous situation of human cancers.
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). Thus, we tested whether a neutralizing IL6 antibody could inhibit the tumor growth of the RasG12V-transformed human kidney cells. We first confirmed that such an antibody effectively neutralized IL6 in a biological assay. Addition of the neutralizing IL6 antibody to culture medium reduced the IL6-dependant growth of B9 cells to the same level as cells that were not induced by IL6 (Fig. 4B). Next, mice pretreated with either the IL6-neutralizing antibody or a control IgG antibody were injected with RasG12V-transformed human kidney cells, and then treated again every 3 d by intratumor injections of either the anti-IL6 or control antibody. Tumors in mice treated with the control IgG antibody first appeared at week 2 and reached maximum tumor volume by day 23. This growth was retarded in the IL6-neutralizing antibody treatment group, and tumors were approximately half the size (P < 0.01) of control tumors (Fig. 4C,D). Consistent with these data, anti-IL6 antibodies have been reported to reduce the tumor growth of prostate cancer cells (Smith and Keller 2001; Wallner et al. 2006). These experiments suggest the intriguing possibility that IL6-neutralizing antibodies may have therapeutic value in the treatment of Ras-driven cancers.
Summary
We show that oncogenic Ras induces the secretion of the cytokine IL6 from a variety of cell types, and that knockdown or genetic ablation of IL6 thwarts Ras-mediated tumor growth of human tumor cell lines and chemical carcinogen-induced tumors. IL6 is thus a bona fide downstream effector of oncogenic Ras that promotes the tumorigenic effects of Ras. IL6 can also act upstream of Ras (Rowley and Van Ness 2002) and, correspondingly, the tumor-promoting effects of IL6 can be context dependant. Nevertheless, in the case of the tested oncogenic Ras-driven tumor cells, IL6 acts downstream from Ras in a paracrine fashion to promote angiogenesis. Overexpression of oncogenic Ras in the tumorigenic HeLa cancer cell line also induced the secretion of a different cytokine IL8, and inhibiting IL8 reduced tumor growth of these cells and the number of CD31-positive cells in a tumor (Sparmann and Bar-Sagi 2004). Like IL6, IL8 was also secreted upon expression of Ras in different human cell types (Supplementary Fig. 3). Thus, Ras-induced secretion of multiple cytokines may underlie the ability of this oncogene to potently induce angiogenesis. Secreted proteins that promote angiogenesis have been successfully inhibited with neutralizing antibodies with desirable clinical outcomes in the treatment of many different human cancers (Adams and Weiner 2005). Consistent with this promising therapeutic strategy to target angiogenesis, and the dependence of oncogenic Ras-driven tumorigenesis on IL6 for angiogenesis, we demonstrate that an IL6-neutralizing antibody inhibits Ras-driven tumor growth. IL6 is thus a druggable protein that holds promise as a target for the treatment of Ras-driven cancers.
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Materials and methods |
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pBabepuro, pBabepuro-H-RasG12V, and pBabepuro-ER:RasG12V were previously described (Lim and Counter 2005). IL6 shRNA-1, shRNA-2, and shRNA-3, and scramble control sequences (5'-AGATGGATGCTTC CAATCTGG-3', 5'-AAGGCAAAGAATCTAGATGCA-3', 5'-AGACAT GTAACAAGAGTAA-3', and 5'-AGACGGAGGCTTACAGTCTGG-3', respectively) were cloned into pSUPER-RETRO-PURO.
Cell lines
Human (embryonic) kidney cells, BJ fibroblasts, mammary epithelial cells, and skeletal muscle myoblasts stably expressing the early region of SV40 (which produce the proteins T-Ag and t-Ag); hTERT; and either H-RasG12V or no transgene (O’Hayer and Counter 2006) were stably infected with retroviruses generated from the indicated vectors to generate polyclonal populations as previously described (O’Hayer and Counter 2006). The ER:RasG12V-expressing tumor kidney cells also expressed p110-CAAX for unrelated reasons (Lim and Counter 2005). B9 cells (Aarden et al. 1987) were a kind gift of Peter Lansdorp (University of British Columbia, Vancouver, BC, Canada). U266 cells were obtained from American Type Culture Collection. HMVECs were a kind gift of Xiao-Fan Wang (Duke University Medical Center, Durham, NC).
RT–PCR
Total RNA was isolated, reverse-transcribed with an oligodT primer, and PCR-amplified with the primers 5'-ATGTAGCCGCCCCACACAGA-3' and 5'-CATCCATCTTTTTCAGCCAT-3' to detect IL6, and 5'-GAAG GTGAAGGTCGGAGACAA-3' and 5'-GCAGAGGGGGCAGAGATGA T-3' to detect GAPDH, using a previously described protocol (Hamad et al. 2002). Cycle number varied between 25 and 40 cycles, depending on cell type and transcript.
Immunoblot
Lysates from the described cell lines were immunoblotted with the primary antibody 
-pan-Ras (Oncogene) or -actin C-2 (Santa Cruz Biotechnology) using standard methods.
IL6R detection
Cells (2 x 106) were incubated with 20 µL of IL6R FITC-conjugated antibody (Abcam) in 3% BSA-PBS for 1 h at 4°C. Cells were then washed three times in PBS and IL6R-positive cells were detected by flow cytometry.
ELISA
Cells were plated at 80% confluency. Twenty-four hours later, cells were washed three times with PBS and cultured in serum-free medium, and 48 h later, cells were collected and analyzed in duplicate with a human IL6 or IL8 ELISA Kit (R&D Systems). Results are reported as means ± standard deviation.
Cell proliferation
Cells (1 x 104 per 6-cm dish) were seeded in triplicate, and viable trypan blue-negative cells were counted daily for 5 d. To measure cell proliferation rate under stress conditions, cells were plated at varying densities in a 96-well plate. Twenty-four hours later, medium was replaced with serum-free medium. Four days later, 50 µL of 5 mg/mL 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma) was added to each well, and 4 h later, medium was aspirated and cells were resuspended in 200 µL of DMSO. Absorbencies were recorded at 540 nm. IL6 antibody neutralization was assayed by seeding 3 x 104 B9 cells per well in a 96-well plate in medium supplemented with 10 pg/mL human IL6 and 0.04 ug/mL monoclonal anti-human IL6 antibody (R&D Systems), which was preincubated to allow binding of the antibody to the cytokine. Forty-eight hours later, 10 µL of 5 mg/mL MTT was added per well. Four hours later, 50 µL of 20% SDS/50% DMF was added per well, and 24 h later, the absorbencies at 570–650 nm were measured.
Soft agar
Of the indicated cells, 5 x 104 cells were suspended in soft agar in triplicate three times independently, and colonies >30 cells were scored after 3 wk, as previously described (Cifone and Fidler 1980; Hamad et al. 2002).
Tumor growth
Cells (1 x 107) mixed with Matrigel were injected subcutaneously into one or both flanks of SCID/beige mice for a total of four injections per cell line, after which tumor volumes were determined at regular intervals as described previously (Hamad et al. 2002). In cases in which tumor-derived cells were retested for IL6 levels, tumors were established in culture under hygromycin selection to enrich for tumor cells as previously described (Lim and Counter 2005). For antibody neutralization of IL6, four animals were pretreated with an injection of 100 µg per mouse IL6-neutralizing antibody (MAB206; R&D Systems) or isotype control antibody (R&D Systems) 2 d prior to tumor cell injection. Starting 1 d after tumor cell injection, mice were treated with neutralizing IL6 antibody or control antibody every 3 d at the tumor site. For chemical carcinogenesis, the backs of 15 control (IL6+/+) C57BL/6J mice and 15 experimental (IL6–/–) C57BL/6J mice, in which both alleles of IL6 were disrupted (B6.129S2-IL6tm1Kopf/J) (Kopf et al. 1994) (Jackson Laboratory) were shaved, and the following day, 150 µL of 125 µg/mL DMBA (Sigma) in DMSO were applied topically, followed 1 wk later by twice-weekly topical applications of 150 µL of 10–4 M TPA (Sigma) in DMSO for 20 wk. One IL6+/+ mouse died at week 11 for unrelated reasons and was excluded from analysis. Tumor number and size were recorded weekly. Student’s t-test was used to compare tumor growth in the various models. The differences between means were considered significant if P < 0.05. All procedures with mice were done under an Institutional Animal Care and Use Committee-approved protocol.
Immunohistochemistry
Excised tumors were fixed in formalin and sectioned. H&E staining, Ki67 (Ki-67 Ab, Zymed), phospho-histone H3 ( phospho-Histone H3 [Ser10] Ab; Upstate Biotechnology), and CD31 (-PECAM-1 Ab; Santa Cruz Biotechnology) immunohistochemistry, and TUNEL assay (Apoptag Plus Peroxidase Apoptosis Detection Kit; Chemicon) were preformed using standard protocols.
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E-MAIL count004{at}mc.duke.edu; FAX (919) 684-8958. 
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1549407
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