Sex specific function of epithelial STAT3 signaling in pathogenesis of K-ras mutant lung cancer

Abstract

Lung adenocarcinomas (LUADs) with mutations in the K-ras oncogene display dismal prognosis. Proinflammatory and immunomodulatory events that drive development of K-ras mutant LUAD are poorly understood. Here, we develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout (LR/Stat3^Δ/Δ) mouse model. Epithelial Stat3 deletion results in intriguing sex-associated discrepancies; K-ras mutant tumors are decreased in female LR/Stat3^Δ/Δ mice whereas tumor burdens are increased in males. RNA-sequencing and tumor microenvironment (TME) analysis demonstrate increased anti-tumor immune responses following Stat3 deletion in females and, conversely, elevated pro-tumor immune pathways in males. While IL-6 blockade in male LR/Stat3^Δ/Δ mice reduces lung tumorigenesis, inhibition of estrogen receptor signaling in female mice augments K-ras mutant oncogenesis and reprograms lung TME toward a pro-tumor phenotype. Our data underscore a critical sex-specific role for epithelial Stat3 signaling in K-ras mutant LUAD, thus paving the way for developing personalized (e.g. sex-based) immunotherapeutic strategies for this fatal disease.

Fig. 1

Epithelial Stat3 deletion induces sex-associated differences in K-ras mutant tumor burden. Lung surface tumor number (left panel) and histopathologic appearance (40× magnification, scale bar = 100 μm) of the lung in female (red circles, n = 12–14) (a) and male (blue squares, n = 12–15) (c) CC-LR (middle panel) and LR/Stat3^Δ/Δ (right panel) mice at the age of 14 weeks. Quantitative analysis (left panel) and representative lung photomicrographs (20× magnification, scale bar = 50 μm) of positive tumor cells for Ki-67 in female (n = 11–14) (b) and male (n = 12–14) (d) CC-LR (middle panel) and LR/Stat3^Δ/Δ (right panel) mice at the age of 14 weeks. (Data represent means ± standard error of mean (SEM); ^****P < 0.0001; ^***P < 0.001; ^**P < 0.005 using two-tailed t-test, experimental replicate # 3–4)

Fig. 2

Sex-differential immune expression programs in lungs of epithelial-specific K-ras mutant Stat3 deficient mice. a Whole-transcriptome sequencing of whole lungs from 14-week-old CC-LR and LR/Stat3^Δ/Δ male and female mice (n = 3 within each genotype and sex group; n = 12 total) was performed using the Ion Torrent Proton platform and as described in the Methods section. Differentially expressed transcripts (n = 339) were identified using a mixed-effects model as described in the Methods section and analyzed by clustering. Rows represent gene features and columns denote samples (yellow, upregulated compared to median sampled; blue, downregulated expression). b Differentially expressed transcripts were functionally and topologically analyzed by pathways and gene set analysis using IPA as described in the Methods section. The gene–gene interaction networks depict significantly predicted inhibition of leukocyte migration and associated gene sets in Stat3-deleted males with the opposite pattern in the female counterparts (orange, activated molecular function; blue, inhibited molecular function; red, upregulated expression; green, downregulated expression)

Fig. 3

Epithelial Stat3 deletion changes the lung tumor microenvironment in a sex-dependent manner. Total inflammatory cell and lineage-specific leukocyte numbers from bronchoalveolar lavage fluid (BALF) of female (n = 7) (a) and male (n = 5–7) (c) CC-LR and LR/Stat3^Δ/Δ mice at the age of 14 weeks. Relative expression of Arg1, Ido, Cxcl1, Ccl2, Il6, Tgfb, Il17, Tnfa, Ifng and Gzmb mRNA in whole lungs of female (b) and male (d) CC-LR and LR/Stat3^Δ/Δ mice at the age of 14 weeks, normalized by CD45 expression (data represent means ± SEM; ^***P < 0.001; ^**P < 0.005; ^*P < 0.05 using two-tailed t-test, experimental replicate # 2-3). WBC, white blood cell

Fig. 4

Neutrophil depletion inhibits K-ras mutant lung tumors in male LR/Stat3^Δ/Δ mice. a Lung surface tumor number in 14-week-old male LR/Stat3^Δ/Δ mice treated with control (IgG2) (n = 4) or anti-Ly6G (n = 4) antibody. b Total inflammatory cell and lineage-specific leukocyte numbers from BALF of control (n = 4) or anti-Ly6G antibody treated (n = 4) male LR/Stat3^Δ/Δ mice. c Relative expression of Arg1, Ido, Cxcl1, Ccl2, Il6, Tgfb, Il17, Tnfa, Ifng and Gzmb mRNA in whole lungs, normalized by CD45 expression, of LR/Stat3^Δ/Δ male mice treated with control (n = 4) or anti-Ly6G (n = 4) antibody (data represent means ± SEM; ^***P < 0.001; ^**P < 0.005; ^*P < 0.05 using two-tailed t-test, experimental replicate # 2-3). WBC, white blood cells

Fig. 5

IL-6 blockade reduces K-ras-induced lung tumorigenesis in male LR/Stat3^Δ/Δ mice. a Lung surface tumor number in 14-week-old male LR/Stat3^Δ/Δ mice after treatment with control (IgG1) (n = 5) or anti-IL-6 (n = 3) antibody. b Total inflammatory cell and lineage-specific leukocyte numbers from BALF of control (n = 5) or anti-IL-6-treated (n = 3) male LR/Stat3^Δ/Δ mice. c Relative expression of Arg1, Ido, Cxcl1, Ccl2, Il6, Tgfb, Il17, Tnfa, Ifng, Foxp3, Tbx21, and Gzmb mRNA in whole lungs, normalized by CD45 expression, of male LR/Stat3^Δ/Δ mice after treatment with control (n = 5) or anti-IL-6 (n = 3) antibody (data represent means ± SEM; ^**P < 0.005, ^*P < 0.05 using two-tailed t-test, experimental replicate # 2–3)

Fig. 6

Estrogen receptor blockade promotes lung tumorigenesis in female LR/Stat3^Δ/Δ mice. a Lung surface tumor number in 14-week-old female LR/Stat3^Δ/Δ mice after corn oil (n = 8) or tamoxifen (n = 3) treatment. b Total inflammatory cell and lineage-specific leukocyte numbers from BALF of female LR/Stat3^Δ/Δ mice after corn oil (n = 4) or tamoxifen (n = 3) treatment. c Relative expression of Arg1, Ido, Ccl2, Il6, Tgfb, Il17, Tnfa, Ifng, Tbx21, Foxp3, and Gzmb mRNA in whole lungs, normalized by CD45 expression, of female LR/Stat3^Δ/Δ mice after corn oil (n = 4) or tamoxifen (n = 3) treatment (data represent means ± SEM; ^**P < 0.005, ^*P < 0.05 using two-tailed t-test, experimental replicate # 2-3)

Fig. 7

Mechanisms underlying sex-associated function of epithelial STAT3 signaling in K-ras mutant lung cancer. Our findings suggest that there is a differential regulation of NF-κB activation in K-ras mutant STAT3-deficient lung epithelium within sex, and estrogen signaling plays a protective role in females by reducing the activation of NF-κB pathway and subsequent induction of an anti-tumor immune response. It also suggests that activation of NF-κB pathway in the lung of male mice drives the tumor-promoting effect of STAT3 deficiency probably by induction of IL-6 and CXCL1, and subsequent recruitment of neutrophils and induction of a pro-tumor immunosuppressive response