Autocrine effects of tumor-derived complement

Abstract

We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications.

Figure 1. Ovarian Cancer Cells Secrete Complement Proteins, which Enhance Tumor Growth

(A) We measured total tumor weight in an orthotopic murine model of ovarian cancer induced by ID8-VEGF murine ovarian cancer cells in C3^−/− and WT control mice, both in C57BL/6 background. n.s, not significant. (B) Immunostaining of tumors induced by ID8-VEGF in WT and C3^−/− mice, using anti-C3 antibody compared to negative control stain (secondary antibody alone). Scale bar length is 100 μm. (C) Quantitative real-time PCR for C3 mRNA on RNA isolated from murine and human ovarian cancer cell lines. Expression of C3 mRNA in cancer cell lines was compared to that in MOECs and normal human ovarian surface epithelial cell lines (HIO 180) (n = 3; **p ≤ 0.01, t test). (D) C3 gene knockdown in SKOV3ip1 human ovarian cancer cells using C3 siRNA, reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (**p ≤ 0.01, t test). (E) C3 gene knockdown in SKOV3ip1-induced tumors by intraperitoneal injection of hC3 siRNA into tumor-bearing NU/NU mice reduced total weight (*p = 0.017) and number of tumor nodules (*p = 0.05). (F) Representative immunostaining for C3, Ki67, and CD31 in tumors resected from hC3 siRNA-injected and scrambled siRNA-injected mice. Scale bar, 100 μm. (G) The proliferation index in resected tumors was quantified as the percentage of Ki67 positivity shown in dot plots (39% in C3 siRNA versus 74% in scrambled siRNA, n = 5 mice in each group; *p = 0.05, t test). The number of blood vessels in resected tumors was quantified by counting the number of CD31+ lumen structures in five high-power fields (HPFs) per section and in five sections per tumor nodule and in five mice per group. Average number of CD31+ lumens per HPF is shown as dot plots (22/HPF in C3 siRNA versus 42/HPF in scrambled siRNA; *p = 0.05, t test). (H) We investigated the effect of complement on proliferation of endothelial cells by measuring the proliferation rate of RF24 endothelial cells after transfection with C3 siRNA. C3 knockdown did not reduce the proliferation rate in RF24 endothelial cells (n = 3; p = 0.07, t test).

Figure 2. C5 Knockdown Decreases Tumor Growth

(A) Silencing of C5 gene expression in SKOV3ip1 cells using hC5 siRNA reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (**p ≤ 0.01, t test). (B) C5 gene knockdown using intraperitoneal injection of hC5 siRNA into SKOV3ip1-induced tumor-bearing NU/NU mice reduced total tumor weight compared to the same mice injected with scrambled siRNA. *p ≤ 0.05. (C) Representative immunostaining for C5 and Ki67 in tumors resected from hC5 siRNA-injected and scrambled siRNA-injected mice. Scale bar, 100 μm. (D) The proliferation index in resected tumors was quantified as the percentage of Ki67 positivity in bar graphs (37.1% in C5 siRNA versus 72.3% in scrambled siRNA, n = 5 in each group; *p = 0.05, t test).

Figure 3. Role of Immunomodulatory Effect of C3 Knockdown on Tumor Growth

(A) ID8-VEGF murine ovarian cancer cells stably expressing murine (m) C3 shRNA or control scrambled shRNA were injected into immune-competent C57BL/6 mice (WT and C3^−/−). Cancer cells with C3 knockdown generated smaller tumors in both WT and C3^−/− mice. **p ≤ 0.0001. (B) Representative immunostaining for CD8 and CD11b in tumors induced by mC3 shRNA-expressing or scrambled shRNA-expressing ID8-VEGF cells in WT mice. Scale bar, 100 μm. (C) The number of cytotoxic T cells (CD8+) and myeloid cells (CD11b+) were determined by counting cells in three high-power fields per tumor nodule per mouse and in five mice per group, and the results are summarized as bar graphs (28 CD8+ cells/HPF in scrambled shRNA-expressing and 358 CD8+/HPF in C3 shRNA-expressing cell-induced tumors; 82 CD11b+ cells/HPF in scrambled shRNA-expressing and average number of 25 CD11b+ cells/HPF in C3 shRNA-expressing cell-induced tumors, n = 15 HPF; *p = 0.001, t test). (D) ID8-VEGF cells stably expressing mC3 shRNA or control scrambled shRNA were injected to CD8^−/− and WT mice. There was no significant difference in tumor size after injection with scrambled shRNA-expressing cancer cells between CD8^−/− and WT mice (0.60 ± 0.23 g versus 0.77 ± 0.17 g, respectively; n = 10 mice/ group; p = 0.19). C3 silencing by mC3 shRNA in cancer cells resulted in 98% reduction in tumor weight in both WT and CD8^−/− mice (*p ≤ 0.001). (E) A representative necropsy in mice showing the presence of tumor nodules on viscera and peritoneal surfaces (surrounded by dashed lines) in WT and CD8^−/− mice injected with ID8-VEGF cells expressing scrambled shRNA and lack of tumor nodules in those injected with mC3 shRNA-expressing cancer cells. Arrows show tumor nodules on the peritoneal surface of diaphragm.

Figure 4. Direct Effect of Complement Activation on Tumor Growth

(A) Agonist peptides to anaphylatoxin receptors (C3aR-AG and C5aR-AG) increase proliferation, migration, and invasion of SKOV3ip1 human ovarian cancer cells in vitro compared to those in cells incubated with scrambled peptide (control). SKOV3ip1 cells were incubated with 0.1 μM C3aR-AG and C5aR-AG for 48 hr (proliferation),6 hr (migration),or24 hr (invasion). Results of three independentexperiments (each of them in triplicate) are summarized as bar graphs(*p ≤0.01,t test). (B) C3aR inhibitor (W-54011), labeled as C3aR-AT, and C5aR inhibitor (SB290157), labeled as C5AR-AT, reduced proliferation of SKOV3ip1 human ovarian cancer cells in vitro compared to those in cells incubated with scrambled peptide (control). SKOV3ip1 cells were incubated with 0.1 μM C3aR-AT and C5aR-AT for 48 hr. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (*p ≤ 0.001). (C) SKOV3ip1 human ovarian cancer cells were incubated with Compstatin (Tocris Bioscience), a peptide inhibitor of C3 convertase, at a final concentration of 150 μg/ml or with the controlled scrambled peptide for 2 days. Cell proliferation was measured at the end of the incubation period using EdU incorporation assay. Compstatin reduced the proliferation rate in SKOV3ip1 cells by 15% (n = 3; *p ≤ 0.002, t test).

Figure 5. Cancer Cells Originated from Different Organs Secrete Complement Proteins that Enhance Cell Proliferation

(A) Quantification of C3 mRNA in h breast, ovarian, lung, and endometrium cancer cell lines using quantitative real-time PCR (n = 3). (B and C) Reducing C3 gene expression in (B) H226 human squamous cell lung cancer and in (C) Hec265 human endometrium cancer cell lines using C3 siRNA reduced proliferation, migration, and invasion of these cells in vitro. Results of three independent experiments (each of them in triplicate) are summarized as bar graphs (*p ≤ 0.01 and **p ≤ 0.001, t test). (D) The relative abundance of AKT mRNA in total mRNA isolated from SKOV3ip1 cells after 24 hr exposure to C3aR and C5aR agonists was quantified by quantitative real-time PCR, and the result of three experiments are summarized as bar graphs (*p ≤ 0.001). (E) A representative immunoblot on total cell lysate prepared from SKOV3ip1 cells after 48 hr exposure to C3aR and C5aR agonists and inhibitors using antibodies to p-85, AKT, and their phosphorylated forms (n = 3). (F) Immunostaining of tumors induced by ID8-VEGF cells expressing scrambled shRNA or mC3 shRNA in C57BL/6 mice using anti-pAKT antibody. (G) To investigate the effect of AKT or PI3K silencing on C3aR and C5aR agonist-induced enhancement of SKOV3ip1 proliferation, expression of AKT or PI3K in SKOV3ip1 was reduced using AKT or PI3K siRNA (data not shown), and cell proliferation was quantified 48 hr after exposure to C3aR-AG and C5aR-AG and was compared to SKOV3ip1 cells transfected with scrambled siRNA (n = 3; *p ≤ 0.001). (H) C3 mRNA level in the tumor specimens of 75 patients diagnosed with ovarian cancer in MDACC was determined using quantitative real-time PCR and correlated with their OS (p = 0.004) and presented as Kaplan-Meier survival curve. (I) Correlation between expression of C5aR in tumor and OS in 562 patients with ovarian cancer in TCGA database (p = 0.019). (J) The amount of C5aR mRNA in tumors resected from 167 patients with a diagnosis of lung squamous cell carcinoma was quantified using quantitative real-time PCR and was correlated to the OS of these patients as documented in TCGA database. The results are shown as Kaplan-Meier survival curve (p = 0.026).

Figure 6. Cancer Cell-Produced Complement Enhances Tumor Growth in an Autocrine Manner

Cancer cells secrete complement proteins resulting in complement activation in the tumor microenvironment. Complement activation products such as C3a and C5a activate their receptors on cancer cells that through PI3K/AKT signaling increase cell proliferation.