Selective neutralization of IL-12 p40 monomer inducesdeath in prostate cancer cells via IL-12–IFN-γMadhuchhanda 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 BrownVeterans 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 reviewApril 12, 2017)

Cancer cells are adept at evading cell death, but the underlyingmechanisms are poorly understood. IL-12 plays a critical role in theearly inflammatory response to infection and in the generation ofT-helper type 1 cells, favoring cell-mediated immunity. IL-12 is com-posed of two different subunits, p40 and p35. This study underlinesthe importance of IL-12 p40 monomer (p40) in helping cancer cells toescape cell death. We found that different mouse and human cancercells produced greater levels of p40 than p40 homodimer (p402),IL-12, or IL-23. Similarly, the serum level of p40 was much greaterin patients with prostate cancer than in healthy control subjects.Selective neutralization of p40, but not p402, by mAb stimulateddeath in different cancer cells in vitro and in vivo in a tumor model.Interestingly, p40was involved in the arrest of IL-12 receptor (IL-12R)IL-12Rβ1, but not IL-12Rβ2, in the membrane, and that p40 neutral-ization induced the internalization of IL-12Rβ1 via caveolin andcaused cancer cell death via the IL-12–IFN-γ pathway. These studiesidentify a role of p40 monomer in helping cancer cells to escape celldeath via suppression of IL-12Rβ1 internalization.

prostate cancer | IL-12 p40 monomer | IL-12Rb1 | IL-12 | IFN-γ

Understanding the mechanisms by which cancer cells escapedeath is an important area of research. Because IL-12 is an

important molecule in terms of cell-mediated immunity (1), thiscytokine is always under scanner for the treatment of cancer (2, 3).The IL-12 family of cytokines has four members, including p40monomer (p40), p40 homodimer (p402), IL-12 (p40:p35), and IL-23(p40:p19). In this era of science dominated by heterodimers, only IL-23 and IL-12 were thought to have biological functions. As a result,p40 and p402 were considered as nonfunctional members of the IL-12 family (4). However, we have demonstrated a proinflammatoryproperty of p402 (5–7) and delineated that biological functions ofp402 are different from those of IL-12 and IL-23 (8, 9). Furthermore,after raising separate functional blocking mAb and ELISA againstmouse p402 and p40 (10), we have delineated that mAb against p402protects mice against experimental allergic encephalomyelitis (11).Here, we demonstrate that different forms of cancer cells, excluding

lung cancer, are associated with specific elevation of p40. A selectiveincrease of p40 was also seen in serum from patients with prostatecancer compared with age-matched controls. Interestingly, selectiveablation of p40 by mAb stimulated cell death in different cancer cellsand in vivo in transgenic adenocarcinoma of the mouse prostate(TRAMP) tumor tissue. Furthermore, p40 suppressed the caveolin-mediated internalization of IL-12 receptor (IL-12R) IL-12Rβ1 andassociated IL-12 signaling in TRAMP tumor cells, and these wereneutralized by p40 mAb. These results delineate a pathogenic roleof p40 whereby it helps cancer cells to evade cell death.

ResultsLevels of IL-12 Family of Cytokines (p40, p402, IL-12, and IL-23) inDifferent Mouse and Human Cancer Cell Lines. First, we monitoredthe levels of cytokines in different cancer cell lines. It was notpossible to examine the role of p40 and p402 in the pathogenesis ofany disease as a result of the unavailability of specific functionalblocking mAbs. Therefore, we generated neutralizing mAbs against

p40 and p402 and developed an ELISA to monitor these cytokinesseparately (10). Mouse squamous (KLN), prostate (TRAMP),breast (4T1), and liver hepatoma (Hepa) cells were cultured underserum-free condition for 48 h, followed by measurement of thelevels of p40, p402, IL-12, and IL-23 by sandwich ELISA. In gen-eral, the levels of IL-12 and IL-23 were very low compared withp40 and p402 in each of these cell lines (Fig. 1 A–C). Interestingly,the level of p40 was much higher than the levels of p402, IL-12, orIL-23 in TRAMP, 4T1, and Hepa cells (Fig. 1 A–C). However,levels of p40 and p402 were almost the same in KLN cells (Fig. 1A),suggesting the specificity of our finding. To confirm the presence ofp40 in cancer cells, we adopted different techniques. First, wemonitored the level of p40 in the supernatants of TRAMP cells bynative PAGE followed by Coomassie staining and found the pres-ence of a 40-kDa protein as the major secretory molecule (Fig. 1D).Second, immunoblot analysis of supernatants of TRAMP cells withour p40 monomer mAb a3-3a showed the presence of p40 insupernatants (Fig. 1E). Finally, intracellular FACS analyses withp40 mAb a3-3a and p402 mAb a3-1d after live/dead cell exclusion(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).Next, we measured the level of p40 in different human cancer

cell lines. Native PAGE analyses followed by Coomassie stainingof supernatants demonstrated that human hepatoma Hep3B(Fig. 1H), prostate LNCaP (Fig. 1I), and breast MCF-7 (Fig. 1J)cancer cells produced significant levels of p40. ELISA ofsupernatants clearly indicated that LNCaP, MCF-7, and Hep3Bcells expressed significantly higher levels of p40 than p402 (Fig.1K). Levels of IL-12 and IL-23 (Fig. 1L) were also much lowercompared with p40 (Fig. 1K). Furthermore, immunoblot analyses of

Significance

Different mouse and human cancer cells produce excess p40monomer compared with p40 homodimer, IL-12, and IL-23. Theserum level of p40 monomer is also much higher in patientswith prostate cancer than in healthy control subjects. Inter-estingly, this p40 monomer helps cancer cells to escape IL-12–IFN-γ–mediated death via suppression of IL-12 receptor-β1internalization. This report demonstrates a biological role ofp40 monomer, the so-called nonfunctional member of theIL-12 family of cytokines, in cancer cell survival. These resultsalso delineate that neutralization of p40 monomer may be atherapeutic target in prostate and other cancers, which areassociated with excessive production of p40.

Author contributions: K.P. designed research; M.K. and A.R. performed research; M.K.and A.R. contributed new reagents/analytic tools; M.K., A.R., and K.P. analyzed data; andM.K. and K.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. X.M. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1To whom correspondence should be addressed. Email: kalipada_pahan@rush.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1705536114/-/DCSupplemental.

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different supernatants with anti-human pan-IL-12p40/p70 antibodyalso showed that all three human cancer cells produced a higherlevel of p40 compared with other IL-12 cytokines (Fig. 1M).

Levels of p40, p402, and IL-12 in Serum of Patients with ProstateCancer. To understand the significance of our finding, we mea-sured levels of p40, p402, and IL-12 in the serum of patients withprostate cancer (n = 11) and healthy control subjects (n = 10).There was no significant difference between patients with pros-tate cancer and healthy controls in terms of age and race (Table1), but the level of p40 was significantly higher [F1,18 = 74.8235(>Fc = 4.42); P < 0.0001 (P = 0.0000000792)] in the serum ofpatients with prostate cancer compared with healthy controls(Table 1 and SI Appendix, Fig. S2A). In contrast, the level of p402was significantly higher [F1,18 = 43.4154 (>Fc = 4.42); P < 0.0005(P = 0.0000345)] in healthy controls relative to prostate cancercases (Table 1 and SI Appendix, Fig. S2B). On the contrary, wedid not observe any significant difference in IL-12 levels [F1,18 =

3.2015 (<Fc = 4.42); P > 0.05 (P = 0.0904)] between controls andpatients with cancer (Table 1 and SI Appendix, Fig. S2C).

Stimulation of Death Response in Different Mouse and Human Cancer Cellsby Selective Neutralization of p40 by Monoclonal Antibody. Because,among the IL-12 family members, the level of p40 was the highest inmost cancer cells, we examined its role in survival of cancer cells. It isoften quite straightforward to consider a KO mouse model to in-vestigate the role of a molecule in any disease process. However, wecannot use p40 (−/−) mice in this case because knocking out thep40 gene will knock down IL-12, IL-23, p402, and p40. Therefore, toinvestigate the role of p402 and p40 in the life and death of differentcancer cells, the only feasible approach was to use neutralizing mAbsagainst these molecules. The p40 mAb a3-3a, but not p402 mAba3-1d, increased LDH (lactate dehydrogenase) release (Fig. 2 A–D) and decreased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 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. On thecontrary, p40 mAb had no effect on LDH or MTT in KLN lungcancer cells, indicating the specificity of the effect. To monitor deathof tumor cells from another perspective, we measured calcium influxthrough the t-type calcium channel. Interestingly, treatment of dif-ferent cancer cells with p40, but not p402, mAb displayed a reducedt-type calcium influx in TRAMP (Fig. 2J), 4T1 (Fig. 2K), and Hepa(Fig. 2L) cells. Again, p40 mAb remained unable to modulate t-typecalcium influx in KLN cancer cells (Fig. 2I). Accordingly, TUNEL(Fig. 2M) and Annexin V labeling (Fig. 2N) followed by quantitativeanalyses (Fig. 2 O and P) reinforced that neutralization of p40, butnot p402, stimulated death in TRAMP, 4T1, and Hepa cancer cells.However, p40 mAb had no effect on the apoptosis of KLN cancercells. Similar to mouse cancer cells, neutralization of p40, but notp402, induced cell death in human LNCaP (SI Appendix, Fig. S3 Aand D), MCF-7 (SI Appendix, Fig. S3 B and E), and Hep3B (SIAppendix, Fig. S3 C and F) cancer cells.

Regression of Tumor Growth and Stimulation of Death Response inVivo in Tumor Tissues by Specific Neutralization of p40. Next, weexamined the effect of p40 mAb on tumor size and the death oftumor tissue in vivo when TRAMP cells were grown as a tumor inthe flank of male C57BL/6 mice. When the tumor had reached 0.8–1 mm in size, mice were treated with p40 mAb a3-3a at an i.p. doseof 2 mg/kg body weight twice per week for 2 wk. The tumor size wasrecorded every alternate day. After 2 wk, tumors were labeled withIR dye 800-conjugated 2-deoxy-D-glucose via tail vein injection andthen imaged in a LI-COR Odyssey IR scanner. Interestingly, weobserved that administration of p40 mAb significantly reduced thesize of tumors as evident from whole-animal IR images (Fig. 3A) andimages of excised tumors (Fig. 3B). From the tumor regression curve,it was clear that the size of tumors in the p40 mAb-treated groupdecreased steadily and significantly [F1,7 = 16.78 (>Fc = 5.59); P <0.01 (P = 0.004); Bonferroni post hoc test] compared with the con-trol group (Fig. 3C). On the contrary, control hamster IgG had nosuch effect (Fig. 3C). Next, we monitored apoptosis in these tumortissues. Our TUNEL results clearly showed that the population ofTUNEL-positive cells in the p40 mAb-treated tumors was higherthan in control or IgG-treated tumors (Fig. 3D), suggesting thatneutralization of p40 by p40 mAb is capable of inducing apoptosis intumor tissues. To further confirm this finding, we monitored themRNA expression of different apoptosis-related genes in treated anduntreated tumor tissues by using a custom gene array. Gene array(Fig. 3E) followed by real-time PCR analysis of individual genes (Fig.3F) clearly indicated that p40 mAb treatment significantly elevatedthe expression of apoptosis-related genes such as caspase 3, caspase7, caspase 8, caspase 9, BAD, BID, cytochrome C, BAK, and p53.

Enlargement of Tumor by Supplementation of p40. Because neu-tralization of p40 induced the death of cancer cells and reducedtumor size, we investigated whether supplementation of p40 had

Fig. 1. Levels of IL-12 family of cytokines in different cancer cells. Levels ofp40 and p402 (A), IL-12 (B), and IL-23 (C) were measured in supernatants ofmouse tumor cells (KLN, TRAMP-C2, 4T1, and Hepa) by ELISA. Results are mean ±SD of three different experiments (aP < 0.001 vs. p40 measured in respectivetumor cells). (D) TRAMP-C2 supernatants were passed through a 10-kDa cutcolumn followed by native PAGE and Coomassie blue staining. The p40 bandwasdetected by comparing with pure p40 protein (far left column). (E) Native PAGEimmunoblot analyses of p40 in the supernatants of TRAMP-C2 from three sep-arate experiments. (F) Intracellular FACS assay of p40 and p402 in culturedTRAMP-C2 cells after live/dead cell exclusion. (G) Mean fluorescence intensity(MFI) of intracellular p40 and p402 was calculated (*P < 0.01 vs. p40; NS, notsignificant). Native PAGE followed by Coomassie staining was performed todetect p40 in supernatants of human cancer cells [Hep3B (H); LnCAP (I); MCF-7(J)]. Levels of p40 and p402 (K) and IL-12 and IL-23 (L) were measured in super-natants of Hep3B, LnCAP, andMCF-7 cells by sandwich ELISA. Results are mean ±SD of three different experiments (aP < 0.001 vs. p40 measured in respectivetumor cells). Native PAGE immunoblot analyses of p40 and other IL-12 membersof cytokines (M) in supernatants of Hep3B, LnCAP, and MCF-7 cells.

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the opposite effects. In fact, at a dose of 20 ng/mL, recombinantp40 increased the survival of TRAMP cells as shown by MTT (SIAppendix, Fig. S4A) and LDH release (SI Appendix, Fig. S4B).Accordingly, recombinant p40 significantly increased the size oftumors as evident from whole-animal IR scanning (SI Appendix,Fig. S4C) and images of excised tumors (SI Appendix, Fig. S4D). Itis clear from the tumor growth curve that the size of tumors in thep40-treated group increased significantly [F1,4 = 9.99 (>Fc = 7.7);P < 0.05 (P = 0.03) at 100 ng per mouse and F1,4 = 10.78 (>Fc =7.7); P < 0.05 (P = 0.03) at 200 ng per mouse on week 8] comparedwith the control group (SI Appendix, Fig. S4E). On the contrary,heat-inactivated p40 had no such stimulatory effect [F1,4 = 0.055(<Fc = 7.7); P > 0.05 (P = 0.825); SI Appendix, Fig. S4 C–E].

Induction of IFN-γ in Cultured Tumor Cells and in Vivo in Tumor Tissueby Specific Neutralization of p40. Next, we investigated the mecha-nism by which p40 mAb induced death response in cancer cells.Induction of IFN-γ production is a proven therapeutic strategy toinduce cytotoxicity in cancer cells (12). Although IFN-γ is a T cell-derived cytokine, other cells such as macrophages (13) and epi-thelial cells (14, 15) are also capable of producing IFN-γ. There-fore, we examined if p40 mAb treatment up-regulated IFN-γ inTRAMP cells. Interestingly, we observed that p40 mAb, but notIgG, significantly increased the mRNA expression of IFN-γ inTRAMP cells (Fig. 4A). Although IFN-γ is a Th1 cell cytokine, ourELISA results (Fig. 4B), intracellular FACS analysis (Fig. 4 C andD) after live/dead cell exclusion (SI Appendix, Fig. S5), and im-munocytochemical analysis (Fig. 4E) clearly indicated that p40mAb treatment increased the level of IFN-γ in TRAMP cells. Onthe contrary, recombinant p40 decreased the level of IFN-γ inTRAMP cells (Fig. 4B). The p40 mAb treatment (Fig. 4F) de-creased the level of IL-10, an antiinflammatory cytokine that isknown to support the growth of cancer cells (16). Next, we in-vestigated if the elevated expression of IFN-γ in p40 mAb-treatedTRAMP cells was indeed involved in the cell death. TUNEL (Fig.4G), LDH (Fig. 4H), and MTT (Fig. 4I) assays revealed that IFN-γ-neutralizing antibody abrogated p40 mAb-mediated cell death inTRAMP cells. Other cancer cells, such as Hepa (SI Appendix, Fig.S6 A and B) and 4T1 (SI Appendix, Fig. S6 C and D), also displayed

up-regulated expression of IFN-γ. MTT (SI Appendix, Fig. S7 A andC) and LDH (SI Appendix, Fig. S7 B and D) assays revealed thatp40 mAb treatment induced death in Hepa (SI Appendix, Fig. S7 Aand B) and 4T1 (SI Appendix, Fig. S7 C and D) cells via IFN-γ.We also observed that p40 mAb-treated tumor tissue ex-

pressed more IFN-γ mRNA (SI Appendix, Fig. S8 A–C) andprotein (SI Appendix, Fig. S8 D and E) compared with controland IgG-treated tumors. In contrast, recombinant p40-treatedtumor tissue expressed less IFN-γ protein compared with con-trol (SI Appendix, Fig. S8D). IFN-γ-neutralizing antibody alsoabrogated tumor-suppressing activity of p40 mAb [F1,7 = 13.7(>Fc = 5.59); P < 0.01 (P = 0.004); Bonferroni post hoc test]in vivo in mice (SI Appendix, Fig. S9), suggesting that neutral-ization of p40 inhibits tumor growth via IFN-γ.

Induction of IL-12 Production in Tumor Cells and in Vivo in TumorTissue by Specific Neutralization of p40. The up-regulation ofIFN-γ is achieved by the activation of IL-12 signaling pathway(12). Whereas the p40 mAb increased the level of IL-12, therecombinant p40 decreased the level of IL-12 in TRAMP cells(SI Appendix, Fig. S10A). Similarly, the p40 mAb treatment in-creased the level of IL-12, and p40 treatment decreased the levelof IL-12, in vivo in tumor tissues compared with control and IgGtreatment (SI Appendix, Fig. S10B). Consistent with the in-volvement of the IL-12 signaling pathway in the induction ofIFN-γ (17), neutralization of IL-12 by functional blocking anti-bodies suppressed p40 mAb-induced expression of T-bet andIFN-γ in TRAMP cells (SI Appendix, Fig. S10 C and D) andin vivo in tumor (SI Appendix, Fig. S10 E and F). Furthermore,neutralizing antibodies against IL-12 abrogated p40 mAb-medi-ated death of TRAMP cells as indicated by MTT (SI Appendix,Fig. S11A) and LDH release (SI Appendix, Fig. S11B). Consis-tent with cell culture results, functional blocking antibodiesagainst IL-12 also abrogated the tumor-suppressing activity ofp40 mAb [F1,7 =12.345 (>Fc = 5.59); P < 0.01 (P = 0.009);Bonferroni post hoc test] in vivo in mice (SI Appendix, Fig. S9).These results suggest that neutralization of p40 induces IFN-γand cell death in cancer cells and in vivo in tumor via IL-12signaling pathway.

Table 1. Levels of p40, p402, and IL-12 in serum of patients with prostate cancer and control subjects

Samples Age, y Race/ethnicity Gleason score PSA, ng/mL Cancer stage p40, pg/mL p402, pg/mL IL-12, pg/mL

PC-1 55 White 6–7 13.15 III 3,471 ± 318 ND 4 ± 0.4PC-2 58 Asian 8–9 42.4 III 2,962 ± 732 330 ± 290 4 ± 0.084PC-3 56 White 8–9 28.1 III 4,246 ± 1,272 618 ± 478 6 ± 1.76PC-4 67 White 7 22.48 III 3,337 ± 2,206 514 ± 68 3 ± 0.753PC-5 61 White 7–8 29.0 III 2,799 ± 291 ND 3 ± 2.93PC-6 56 White 8–9 56.4 III 2,494 ± 1,251 454 ± 88 6 ± 2.49PC-7 60 White 8–9 24.6 III 3,590 ± 1,822 ND 3 ± 2.83PC-8 59 Caucasian 7 4 IV 3,446 ± 1,082 ND 4 ± 1.37PC-9 52 White 6 28.4 II 1,490 ± 543 ND 4.9 ± 0.21PC-10 76 Caucasian 9 6.52 IV 3,704 ± 1,460 ND 5 ± 3.81PC-11 71 Caucasian NA 102.79 IV 2,184 ± 751 ND 7 ± 1.61Control-1 65 White Healthy Healthy Healthy 1,060 ± 855 1,164 ± 328 3 ± 0.448Control-2 63 White Healthy Healthy Healthy 1,018 ± 333 729 ± 530 6 ± 0.097Control-3 83 White Healthy Healthy Healthy 866 ± 540 2,361 ± 235 3 ± 0.097Control-4 63 White Healthy Healthy Healthy 863 ± 359 1,991 ± 785 5 ± 0.98Control-5 61 White Healthy Healthy Healthy 1,055 ± 768 1,884 ± 433 6 ± 0.098Control-6 57 Hispanic Healthy Healthy Healthy 1,198 ± 395 2,148 ± 539 10 ± 1.69Control-7 70 White Healthy Healthy Healthy 1,343 ± 728 1,765 ± 507 3 ± 0.53Control-8 67 White Healthy Healthy Healthy 606 ± 401 1,534 ± 634 6 ± 2.05Control-9 66 White Healthy Healthy Healthy 1,003 ± 293 1,192 ± 325 6 ± 1.22Control-10 52 Asian Healthy Healthy Healthy 865 ± 107 626 ± 200 10 ± 4.5

Serum samples were obtained from Discovery Life Sciences. Each sample was analyzed for p40, p402, and IL-12 three times by ELISA. NA, not available; ND,not detectable; p40, p40 monomer; p402, p40 homodimer; PC, prostate cancer.

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Internalization of IL-12Rβ1 in Tumor Cells by Selective Neutralizationof p40. The IL-12 signaling pathway is initiated by the interactionbetween IL-12 and IL-12R, which is a heterodimer of IL-12Rβ1and IL-12Rβ2. A functional IL-12R has been reported to be in-ternalized after successful binding with its ligand IL-12 (18).Therefore, we examined if p40 monomer was involved in the ar-resting of IL-12R on TRAMP cells to negate the IL-12 signalingpathway. Interestingly, our FACS analyses revealed that thetreatment with p40, but not with p402 or p70, increased the surfaceexpression of IL-12Rβ1 in TRAMP cells (Fig. 5A, i–iv). In contrast,p40 did not have any effect on the surface expression of IL-12Rβ2(SI Appendix, Fig. S12). Furthermore, treatment with p40 mAb,but not IgG, down-regulated the membrane level of IL-12Rβ1(Fig. 5A, v and vi), suggesting the involvement of p40 in arrestingIL-12Rβ1 on the membrane. To further confirm, we performedimmunoblot analyses of IL-12Rβ1 in the membrane fraction ofTRAMP cells. Interestingly, we found that treatment with p40, butnot with p402 or IL-12, increased the presence of IL-12Rβ1 in themembrane (Fig. 5B). Pan-cadherin was analyzed to check thepurity of the membrane fraction (Fig. 5C, Top). Surprisingly, wefound increased levels of β-actin in membrane fractions of p402-and p70-treated TRAMP cells, suggesting that the treatment withp402 or p70 is possibly associated with increased formation of

endocytic vesicles in the membrane (Fig. 5C, Bottom). In contrast,we did not observe increased membrane levels of β-actin in p40-treated TRAMP cells (Fig. 5C). These results suggest that p40, butneither p402 nor p70, may be involved in arresting IL-12Rβ1 in themembrane. However, there was no difference in IL-12Rβ1 inwhole-cell extract when TRAMP cells were treated with thesecytokines (Fig. 5D), negating the possibility of induction of IL-12Rβ1 level by p40 monomer. Consistently, the p40 mAb abro-gated p40-mediated increase in IL-12Rβ1 in membrane of TRAMPcells (Fig. 5E), suggesting that p40 is indeed involved in the mem-brane arrest of IL-12Rβ1. Pan-cadherin was analyzed to monitor thepurity of the membrane fraction (Fig. 5F, Top). The level of β-actinwas higher in p40 mAb-treated cells, suggesting that the absence ofp40 may induce the formation of endocytic vesicles in TRAMP cells(Fig. 5F, Bottom). However, again, there was no difference in thelevel of total IL-12Rβ1 between cells treated with p40 with orwithout p40 mAb and cells treated with p40, suggesting that p40mAb treatment does not down-regulate the expression of IL-12Rβ1in TRAMP cells (Fig. 5G). Together, these results suggest thatexcess p40 released by TRAMP cells inhibit IL-12 signaling bysuppressing the internalization or endocytosis of IL-12Rβ1.

Fig. 2. Monoclonal antibody-mediated neutralization of p40, but not p402,induces death of different mouse cancer cells. KLN (A and E), TRAMP-C2 (B andF), 4T1 (C and G), and Hepa (D and H) cells were treated with mAbs againstp40 and p402 for 48 h under serum-free conditions followed by monitoring ofLDH release (A–D) and MTT [(E–H) *P < 0.01 vs. control]. t-type calcium influxwas performed in KLN (I), TRAMP-C2 (J), 4T1 (K), and Hepa (L) cells. t-type cal-cium influx was measured in the presence of 1 M KCl. TUNEL assay (M) andphycoerythrin-tagged Annexin V staining (N) were performed in KLN, TRAMP-C2, 4T1, and Hepa cells. TUNEL-positive (O) and phycoerythrin-tagged AnnexinV-positive (P) cells were counted in 10 different images per group and thenplotted as percentages of control. All results are mean ± SD of three differentexperiments (*P < 0.001 vs. respective controls for TUNEL and Annexin V assays).

Fig. 3. Regression of TRAMP tumor in vivo in mice by p40 mAb. (A) Eight- to10-wk-old male C57BL/6 mice (n = 5 per group) were injected s.c. with asuspension of 1 million TRAMP-C2 cells. After approximately 6 wk, whentumors were 0.8–1 mm in size, mice were treated with p40 mAb (Middle)and hamster IgG (Right) at a dose of 2 mg/kg body weight twice per week.After 2 wk, tumors were labeled with Alexa 800-conjugated 2-deoxy-D-glucose dye via tail vein injection and then imaged in a LI-COR Odyssey IRimaging system. Results were compared with the control group (Left).(B) Tumors were excised from the flank of all groups of mice. Five mice wereincluded in each group. (C) Tumor size was monitored every alternate day.Results are mean ± SEM of five different mice. (D) TUNEL assay in control,IgG, and p40 mAb-treated tumors (green, β-actin; red, TUNEL). (E) CustommRNA array for 12 different apoptotic genes in control and p40 mAb-treated group, which was then plotted with Heat Map Explorer software.(F) Real-time mRNA analyses of 12 apoptotic genes. Results are mean ± SEMof five mice (*P < 0.05 vs. control and **P < 0.01 vs. control).

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Next, we investigated mechanisms by which neutralization ofp40 induced the internalization of IL-12Rβ1. Receptor internalizationprimarily involves clathrin-dependent and caveolin-dependent path-ways. To examine the involvement of clathrin or caveolin, we usedtwo pharmacological inhibitors, filipin and chlorpromazine (cpm),respectively. Interestingly, pretreatment with filipin, but not cpm,significantly inhibited the membrane internalization of IL-12Rβ1 inp40 mAb-treated TRAMP cells (Fig. 5 H and I), suggesting that p40-mediated internalization of IL-12Rβ1 occurs via a caveolin-sensitiveand clathrin-independent pathway. Immunofluorescence analysisfurther confirmed that the p40 mAb-mediated internalization ofIL-12Rβ1 in TRAMP cells is caveolin-dependent (Fig. 5 J–L).

DiscussionIL-12 (p35:p40) is an important cytokine for triggering cell-mediated immune response (4). It is produced mainly by antigen-presenting cells upon activation through Toll-like receptors and byinteractions with CD4+ T cells (4, 19). Eventually, p40 was shownto pair with p19 to form a newly discovered cytokine, IL-23 (20).Either p19 or p35 is constitutively expressed in many cell types. It

is known that dendritic cells and macrophages, cells that are ableto secrete heterodimeric IL-12 or IL-23, always produce an excessof p40 as monomer or homodimer (i.e., p402) (4). However, bi-ological functions of p402 and p40 remained unknown as a resultof the unavailability of specific neutralizing antibodies.Here, we demonstrate that different cancer cells produce ex-

cess p40 compared with p402, IL-12, and IL-23, and that p40 isinvolved in cancer cell survival. Our conclusion is dependent onthe following observations. First, the level of p40 was muchhigher in mouse (TRAMP, 4T1, and Hepa) as well as human(LNCaP, Hep3b, and MCF-7) cancer cells compared with p402,IL-12, and IL-23. On the contrary, we did not notice this selectiveincrease in p40 in KLN mouse lung cancer cells, indicating thespecificity of this finding. Second, serum of patients with prostatecancer also contained greater levels of p40 compared with healthycontrol subjects. Third, neutralization of p40, but not p402, induced

Fig. 4. Role of IFN-γ in p40 mAb-mediated death of TRAMP-C2 cells. (A) Cellswere treated with p40 mAb and control IgG for 6 h, followed by real-time PCRanalysis for IFN-γ mRNA expression (aP < 0.01 vs. control). (B) Levels of IFN-γwere measured in supernatants by ELISA after 24 h of treatment (aP <0.001 and bP < 0.01 vs. control). (C) Intracellular IFN-γ FACS was performedafter live/dead cell exclusion. Phycoerythrin (PE) control was included to showthe specificity. (D) MFI of IFN-γ was calculated. (E) Immunocytochemicalanalyses of IFN-γ (green) in TRAMP cells after 24 h of treatment. Resultsrepresent three independent experiments. (F) Levels of IL-10 were measuredby ELISA after 24 h of treatment (aP < 0.05 vs. control). (G) TUNEL assay incontrol cells and cells treated with p40 mAb, IgG, p40 mAb plus differentdoses of IFN-γ-neutralizing Ab, p40 mAb plus IgG, and IFN-γ Ab TRAMP cellsafter 48 h of treatment. Under similar treatment conditions, LDH (H) and MTT(I) assays were performed. Results are mean ± SD of three different experi-ments (aP < 0.01 vs. control; bP < 0.01 vs. p40 mAb-treated cells).

Fig. 5. Neutralization of p40 by mAb stimulates the internalization of IL-12Rβ1in TRAMP-C2 cells. (A) Cells were treated with p40 (20 ng/mL), p402 (20 ng/mL),p70 (20 ng/mL), p40mAb (0.5 μg/mL), andmouse IgG (0.5 μg/mL) for 2 h in serum-free conditions followed by FACS analyses of IL-12Rβ1 in control cells (i) and cellstreated with p70 (ii), p40 (iii), p402 (iv), p40 mAb (v), and IgG (vi). Immunoblotanalyses of membrane-bound IL-12Rβ1 (B), pan-cadherin (pCAD) (C), and totalIL-12Rβ1 (D) in p40-, p402-, p70-, p40 mAb-, and IgG-treated TRAMP cells. Im-munoblot analyses of membrane-bound IL-12Rβ1 (E), pCAD (F), and totalIL-12Rβ1 (G) in p40 cytokine- and p40 mAb-treated TRAMP cells. Results representthree independent experiments. Immunoblot analyses of IL-12Rβ1 (H) in themembrane fraction of p40 mAb-treated TRAMP cells pretreated with 5 μM filipinor 2 μM cpm for 2 h. Immunoblot results were normalized with pCAD immuno-blot (Bottom). (I) Relative density of immunoblot analyses normalized with pCAD.Results are mean ± SD of three different experiments (*P < 0.001 vs. p40 mAb).Immunocytochemical analyses of IL-12 Rβ1 (red) and caveoiln-1 (Cav-1; green) in(J) control, (K) p40 mAb-, and (L) p40 mAb plus filipin-treated TRAMP cells. Nucleiwere stained with DAPI. (M) Schematic presentation by which neutralization ofp40 induces cell-mediated immunity in TRAMP cells.

11486 | www.pnas.org/cgi/doi/10.1073/pnas.1705536114 Kundu et al.

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death in different mouse and humor cancer cells. Again, p40 mAbhad no effect on the survival of KLN cells. Fourth, t-type calciuminflux, a growth-supportive event in cancer cells, was significantlyreduced in TRAMP, 4T1, and Hepa cells, but not KLN cells, whentreated with p40 neutralizing antibody. Fifth, our TUNEL andAnnexin-V staining experiments displayed more death in TRAMP,4T1, and Hepa cells, but not KLN cells, after treatment withp40 mAb. Finally, i.p. injection of p40 mAb, but not IgG, signifi-cantly reduced the size of the tumors grown in the flank of maleC57BL/6 mice. This report demonstrates a biological role ofp40 monomer in cancer cell survival. As the serum of patients withprostate cancer also contained higher levels of p40 compared withthat of control subjects, these results indicate the possible thera-peutic prospect of p40 neutralization in cancer.While investigating mechanisms behind p40-mediated killing of

tumor cells, we observed up-regulation of IFN-γ, a major cytotoxicinflammatory cytokine (21), by p40 mAb in pure TRAMP cells. Thisobservation is striking, as T lymphocytes (22) and natural killer cells(23) are considered primary sources of IFN-γ. However, previousstudies demonstrated that it can be produced by murine macro-phages (13) as well as epithelial cells (14, 15), prompting us to ex-amine IFN-γ production in TRAMP epithelial cells. We found thatTRAMP cells expressed very little IFN-γ in unstimulated conditionsand that p40 mAb treatment stimulated the production of IFN-γ byseveral fold. Moreover, cancer cells with epithelial origin such as4T1 and hepatocellular origin such as Hepa also expressed significantamounts of IFN-γ when treated with p40 mAb, further suggestingthat functional blocking of p40 could stimulate IFN-γ production in awide range of cancer cells. Consistent with the cytotoxic nature ofIFN-γ, neutralization of this molecule abrogated p40 mAb-mediateddeath of tumor cells and suppression of tumor growth in vivo in mice,demonstrating that p40 mAb induces death of cancer cells via IFN-γ.Activation of the IL-12 signaling pathway induces IFN-γ pro-

duction in various cells via JAK-STAT pathway (4, 24, 25). Ac-cordingly, p40 mAb stimulated the production of IL-12 and IFN-γ in TRAMP cells, suggesting that the absence of p40 may favorthe interaction of IL-12 with IL-12R to turn on the transcription ofIFN-γ (24) in these cells. This is interesting because, in cell-

mediated immunity, IL-12 produced from antigen-presenting cellsstimulates IFN-γ from T cells. In contrast, IL-12 generated fromcancer cells in response to p40 neutralization is inducing IFN-γproduction from the same cells in an autocrine fashion. A successfulinteraction between IL-12 and IL-12R transmits the downstreamsignal and then internalizes the receptor inside the cell. On thecontrary, an unsuccessful interaction between IL-12 and IL-12Rleaves the receptor arrested in the membrane and unable to trans-mit any downstream signal. Interestingly, we have found that p40 isinvolved in the membrane arrest of IL-12Rβ1, but not IL-12Rβ2.

Materials and MethodsAnimals and Regents. SI Appendix, SI Materials and Methods, provides furtherdetails in regard to animals and regents, sandwich ELISA procedures, flowcytometry analysis with live/dead cell exclusion, tumor development andmeasurement, tissue preparation and immunohistochemistry, semiquantitativeRT-PCR and real-time PCR, FACS, and membrane isolation. Animal maintainingand experiments were in accordance with National Institutes of Healthguidelines and were approved by the Institutional Animal Care and Use com-mittee (IACUC 14-019) of the Rush University of Medical Center, Chicago, IL.

MTT and LDH Assays. MTT and LDH assays were performed as described pre-viously (26).

TUNEL Assay. Following treatments with mAb against p40 or p402, TUNELassays were performed as described previously (26).

Immunoblot Analyses. Immunoblot analyses were performed as described pre-viously (26) with the use of different primary antibodies (SI Appendix, Table S1).

Statistical Analysis. For tumor regression, quantitative data were presented asthe mean ± SEM. Statistical significance was accessed via one-way ANOVAwith Student–Newman–Keuls post hoc analysis. Other data were expressedas means ± SD of three independent experiments. Statistical differencesbetween means were calculated by the Student’s t test. A P value of less than0.05 was considered statistically significant.

ACKNOWLEDGMENTS. This study was supported by National Institutes ofHealth Grants NS83054 and NS97426 and Veteran Affairs Merit AwardsI01BX002174 and 1I01BX003033.

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