Prolactin prevents hepatocellular carcinoma by restricting innate immune activation of c-Myc in mice

Abstract

Women are more resistant to hepatocellular carcinoma (HCC) than men despite equal exposure to major risk factors, such as hepatitis B or C virus infection. Female resistance is hormone-dependent, as evidenced by the sharp increase in HCC incidence in postmenopausal women who do not take hormone replacement therapy. In rodent models sex-dimorphic HCC phenotypes are pituitary-dependent, suggesting that sex hormones act via the gonadal-hypophyseal axis. We found that the estrogen-responsive pituitary hormone prolactin (PRL), signaling through hepatocyte-predominant short-form prolactin receptors (PRLR-S), constrained TNF receptor-associated factor (TRAF)-dependent innate immune responses invoked by IL-1β, TNF-α, and LPS/Toll-like receptor 4 (TLR4), but not TRIF-dependent poly(I:C)/TLR3. PRL ubiquitinated and accelerated poststimulatory decay of a "trafasome" comprised of IRAK1, TRAF6, and MAP3K proteins, abrogating downstream activation of c-Myc-interacting pathways, including PI3K/AKT, mTORC1, p38 MAPK, and NF-κB. Consistent with this finding, we documented exaggerated male liver responses to immune stimuli in mice and humans. Tumor promotion through, but regulation above, the level of c-Myc was demonstrated by sex-independent HCC eruption in Alb-Myc transgenic mice. PRL deficiency accelerated liver carcinogenesis in Prl(-/-) mice of both sexes. Conversely, pharmacologic PRL mobilization using the dopamine D2 receptor antagonist domperidone prevented HCC in tumor-prone C3H/HeN males. Viewed together, our results demonstrate that PRL constrains tumor-promoting liver inflammation by inhibiting MAP3K-dependent activation of c-Myc at the level of the trafasome. PRL-targeted therapy may hold promise for reducing the burden of liver cancer in high-risk men and women.

Keywords: innate immunity; lactotrophs; liver neoplasms; sex dimorphism.

Fig. 1.

Prolactin/female sex constrains hepatocellular IL-1β responses in vitro and in vivo. (A) PRL preconditioning in AML12 hepatocytes reduces IL-1β phosphorylation of p38 MAPK, NF-κB p65/RelA, and AKT. (B) IL-1β induction of APR genes is diminished in PRL-preconditioned hepatocytes. (C) Decreased phosphorylation of p38 MAPK, NF-κB p65/RelA, and AKT but not STAT3, JNK, or ERK1/2 in IL-1β–challenged female versus male mouse liver. (D) Muted induction of inflammatory and tumor-associated genes in IL-1β–challenged females versus males. Data are expressed as mean ± SEM (n = 5 per sex); *P < 0.05, Student t test.

Fig. 2.

Adult men without primary liver disease maintain a more proinflammatory hepatic microenvironment than women. (A) Heat map demonstrates clear distinction between male and female human liver (n = 7 per sex). (B) qRT-PCR confirmation of sex-specific expression of selected feminine (pink) and masculine (blue) genes. (C) Adult men exhibit increased expression of inflammatory and proto-oncogenes, including c-Myc. (D) Top sex-specific biological function categories are associated with cancer and inflammation. *P < 0.05, Student t test.

Fig. 3.

PRL inhibits immune induction of MAP3K-dependent pathways including PI3K/AKT/mTORC1 by accelerating degradation of trafasome. (A) PRL constrains mTORC1 members mTOR and 4E-BP1 in parallel with AKT. (B) MAP3K inhibitor 5(Z)-7-oxozeaenol, but not Ras inhibitor manumycin, mirrors PRL in disruption of p38 MAPK, AKT, and NF-κB p65/RelA phosphorylation. (C, Upper) Western blot demonstrates that prior PRL accelerates degradation of IRAK1 following IL-1β challenge in AML12 cells. (Lower) TRAF6 immunoprecipitation (IP) followed by immunoblot (IB) shows nondegrading ubiquitination by PRL alone, and accelerated degradation of IRAK1 and TRAF6 following IL-1β challenge.

Fig. 4.

Female HCC resistance is PRL-dependent and regulated above c-Myc. (A) Absence of sex bias in preneoplastic foci and HCC in Alb-Myc(tg) mice with constitutive hepatic c-Myc expression. (B) Immunohistochemical confirmation of cytoplasmic and nuclear c-Myc overexpression (diaminobenzidine; brown) in transgenic mice of both sexes compared with WT controls (magnification, 400×). (C) Increased HCC incidence (Upper) and multiplicity (Lower) in PRL-deficient mice relative to WT controls. (D) DEN-initiated Prl^−/− mice have greater local (arrowheads) and vascular (arrows) tumor invasion than sex-matched WT controls (magnification, 200×). Data are expressed as mean ± SEM (n = 9–10 per sex per treatment protocol), *P < 0.05, one-way ANOVA with Tukey posttest.

Fig. 5.

PRL-mobilizing DRD2 antagonist domperidone prevents male HCC. (A) α-DRD2 domperidone increases circulating male PRL to near female levels. (B) Domperidone in male mice reduces multiplicity of dysplastic nodules to intermediate levels between untreated males and females. (C) Domperidone markedly reduces male HCC incidence (22% vs. 100% in untreated males). (D) Tumor-associated transcriptional and morphologic biomarkers in α-DRD2–treated males mirror those of females. (E) Gross and histologic appearance of DEN-induced lesions in α-DRD2 males approximate those found in females. F, foci of cellular alteration; H, HCC; 8-oxo-G, 8-oxoguanine; magnification, 100×. Data are represented as mean ± SEM, *P < 0.05 (n = 9–10 mice per sex per treatment protocol), one-way ANOVA with Tukey posttest.

Fig. 6.

Working model of PRL interruption of TRAF/MAP3K-dependent innate immune activation of the proto-oncogene c-Myc via PI3K/AKT/mTORC1, p38 MAPK and/or NF-κB in hepatocytes. Inhibitory pathways shown in red, inflammatory in blue and proliferative in green. Abbreviations: DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern; PRR, pattern-recognition receptor.