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Selective Neutralization of IL-12 P40 Monomer Induces Death in Prostate Cancer Cells Via IL-12–IFN-Γ

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Selective Neutralization of IL-12 P40 Monomer Induces Death in Prostate Cancer Cells Via IL-12–IFN-Γ Selective neutralization of IL-12 p40 monomer induces death in prostate cancer cells via IL-12–IFN-γ Madhuchhanda Kundua, Avik Roya, and Kalipada Pahana,b,1 aDepartment of Neurological Sciences, Rush University Medical Center, Chicago, IL 60612; and bDivision of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612 Edited by Xiaojing Ma, Weill Medical College, New York, NY, and accepted by Editorial Board Member Carl F. Nathan September 18, 2017 (received for review April 12, 2017) Cancer cells are adept at evading cell death, but the underlying p40 and p402 and developed an ELISA to monitor these cytokines mechanisms are poorly understood. IL-12 plays a critical role in the separately (10). Mouse squamous (KLN), prostate (TRAMP), early inflammatory response to infection and in the generation of breast (4T1), and liver hepatoma (Hepa) cells were cultured under T-helper type 1 cells, favoring cell-mediated immunity. IL-12 is com- serum-free condition for 48 h, followed by measurement of the posed of two different subunits, p40 and p35. This study underlines levels of p40, p402, IL-12, and IL-23 by sandwich ELISA. In gen- the importance of IL-12 p40 monomer (p40) in helping cancer cells to eral, the levels of IL-12 and IL-23 were very low compared with escape cell death. We found that different mouse and human cancer p40 and p402 in each of these cell lines (Fig. 1 A–C). Interestingly, cells produced greater levels of p40 than p40 homodimer (p402), the level of p40 was much higher than the levels of p402, IL-12, or IL-12, or IL-23. Similarly, the serum level of p40 was much greater IL-23 in TRAMP, 4T1, and Hepa cells (Fig. 1 A–C). However, in patients with prostate cancer than in healthy control subjects. levels of p40 and p402 were almost the same in KLN cells (Fig. 1A), Selective neutralization of p40, but not p402,bymAbstimulated suggesting the specificity of our finding. To confirm the presence of death in different cancer cells in vitro and in vivo in a tumor model. p40 in cancer cells, we adopted different techniques. First, we Interestingly, p40 was involved in the arrest of IL-12 receptor (IL-12R) monitored the level of p40 in the supernatants of TRAMP cells by IL-12Rβ1, but not IL-12Rβ2, in the membrane, and that p40 neutral- native PAGE followed by Coomassie staining and found the pres- ization induced the internalization of IL-12Rβ1 via caveolin and ence of a 40-kDa protein as the major secretory molecule (Fig. 1D). caused cancer cell death via the IL-12–IFN-γ pathway. These studies Second, immunoblot analysis of supernatants of TRAMP cells with INFLAMMATION identify a role of p40 monomer in helping cancer cells to escape cell our p40 monomer mAb a3-3a showed the presence of p40 in IMMUNOLOGY AND death via suppression of IL-12Rβ1 internalization. supernatants (Fig. 1E). Finally, intracellular FACS analyses with p40 mAb a3-3a and p402 mAb a3-1d after live/dead cell exclusion prostate cancer | IL-12 p40 monomer | IL-12Rb1 | IL-12 | IFN-γ (SI Appendix,Fig.S1) also showed that the level of p40 was sig- nificantly higher than p402 in TRAMP cells (Fig. 1 F and G). nderstanding the mechanisms by which cancer cells escape Next, we measured the level of p40 in different human cancer Udeath is an important area of research. Because IL-12 is an cell lines. Native PAGE analyses followed by Coomassie staining important molecule in terms of cell-mediated immunity (1), this of supernatants demonstrated that human hepatoma Hep3B H I J cytokine is always under scanner for the treatment of cancer (2, 3). (Fig. 1 ), prostate LNCaP (Fig. 1 ), and breast MCF-7 (Fig. 1 ) The IL-12 family of cytokines has four members, including p40 cancer cells produced significant levels of p40. ELISA of supernatants clearly indicated that LNCaP, MCF-7, and Hep3B monomer (p40), p40 homodimer (p402), IL-12 (p40:p35), and IL-23 cells expressed significantly higher levels of p40 than p402 (Fig. (p40:p19). In this era of science dominated by heterodimers, only IL- K L 23andIL-12werethoughttohavebiological functions. As a result, 1 ). Levels of IL-12 and IL-23 (Fig. 1 ) were also much lower compared with p40 (Fig. 1K). Furthermore, immunoblot analyses of p40 and p402 were considered as nonfunctional members of the IL- 12 family (4). However, we have demonstrated a proinflammatory Significance property of p402 (5–7) and delineated that biological functions of p402 are different from those of IL-12 and IL-23 (8, 9). Furthermore, after raising separate functional blocking mAb and ELISA against Different mouse and human cancer cells produce excess p40 mouse p402 and p40 (10), we have delineated that mAb against p402 monomer compared with p40 homodimer, IL-12, and IL-23. The protects mice against experimental allergic encephalomyelitis (11). serum level of p40 monomer is also much higher in patients Here, we demonstrate that different forms of cancer cells, excluding with prostate cancer than in healthy control subjects. Inter- lung cancer, are associated with specific elevation of p40. A selective estingly, this p40 monomer helps cancer cells to escape IL-12– increase of p40 was also seen in serum from patients with prostate IFN-γ–mediated death via suppression of IL-12 receptor-β1 cancer compared with age-matched controls. Interestingly, selective internalization. This report demonstrates a biological role of ablation of p40 by mAb stimulated cell death in different cancer cells p40 monomer, the so-called nonfunctional member of the and in vivo in transgenic adenocarcinoma of the mouse prostate IL-12 family of cytokines, in cancer cell survival. These results (TRAMP) tumor tissue. Furthermore, p40 suppressed the caveolin- also delineate that neutralization of p40 monomer may be a mediated internalization of IL-12 receptor (IL-12R) IL-12Rβ1and therapeutic target in prostate and other cancers, which are associated IL-12 signaling in TRAMP tumor cells, and these were associated with excessive production of p40. neutralized by p40 mAb. These results delineate a pathogenic role Author contributions: K.P. designed research; M.K. and A.R. performed research; M.K. of p40 whereby it helps cancer cells to evade cell death. and A.R. contributed new reagents/analytic tools; M.K., A.R., and K.P. analyzed data; and M.K. and K.P. wrote the paper. Results The authors declare no conflict of interest. Levels of IL-12 Family of Cytokines (p40, p402, IL-12, and IL-23) in This article is a PNAS Direct Submission. X.M. is a guest editor invited by the Different Mouse and Human Cancer Cell Lines. First, we monitored Editorial Board. the levels of cytokines in different cancer cell lines. It was not Published under the PNAS license. possibletoexaminetheroleofp40andp402 in the pathogenesis of 1To whom correspondence should be addressed. Email: [email protected]. any disease as a result of the unavailability of specific functional This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. blocking mAbs. Therefore, we generated neutralizing mAbs against 1073/pnas.1705536114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1705536114 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 3.2015 (<Fc = 4.42); P > 0.05 (P = 0.0904)] between controls and patients with cancer (Table 1 and SI Appendix, Fig. S2C). Stimulation of Death Response in Different Mouse and Human Cancer Cells by Selective Neutralization of p40 by Monoclonal Antibody. Because, among the IL-12 family members, the level of p40 was the highest in most cancer cells, we examined its role in survival of cancer cells. It is often quite straightforward to consider a KO mouse model to in- vestigate the role of a molecule in any disease process. However, we − − cannot use p40 ( / ) mice in this case because knocking out the p40 gene will knock down IL-12, IL-23, p402, and p40. Therefore, to investigate the role of p402 and p40 in the life and death of different cancer cells, the only feasible approach was to use neutralizing mAbs against these molecules. The p40 mAb a3-3a, but not p402 mAb a3-1d, increased LDH (lactate dehydrogenase) release (Fig. 2 A– D) and decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Fig. 2 E–H) in TRAMP (Fig. 2 B and F), 4T1 (Fig. 2 C and G), and Hepa (Fig. 2 D and H)cells.Onthe contrary, p40 mAb had no effect on LDH or MTT in KLN lung cancer cells, indicating the specificity of the effect. To monitor death of tumor cells from another perspective, we measured calcium influx through the t-type calcium channel. Interestingly, treatment of dif- ferent cancer cells with p40, but not p402, mAb displayed a reduced t-type calcium influx in TRAMP (Fig. 2J), 4T1 (Fig. 2K), and Hepa (Fig. 2L) cells. Again, p40 mAb remained unable to modulate t-type calcium influx in KLN cancer cells (Fig. 2I). Accordingly, TUNEL (Fig. 2M) and Annexin V labeling (Fig. 2N) followed by quantitative analyses (Fig. 2 O and P) reinforced that neutralization of p40, but not p402, stimulated death in TRAMP, 4T1, and Hepa cancer cells. However, p40 mAb had no effect on the apoptosis of KLN cancer cells. Similar to mouse cancer cells, neutralization of p40, but not p402, induced cell death in human LNCaP (SI Appendix,Fig.S3A Fig. 1. Levels of IL-12 family of cytokines in different cancer cells. Levels of and D), MCF-7 (SI Appendix,Fig.S3B and E), and Hep3B (SI p40 and p402 (A), IL-12 (B), and IL-23 (C) were measured in supernatants of Appendix,Fig.S3C and F)cancercells.
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