Dysfunctional adipocytes promote tumor progression through YAP/TAZ-dependent cancer-associated adipocyte transformation

Abstract

Obesity has emerged as a prominent risk factor for the development of malignant tumors. However, the existing literature on the role of adipocytes in the tumor microenvironment (TME) to elucidate the correlation between obesity and cancer remains insufficient. Here, we aim to investigate the formation of cancer-associated adipocytes (CAAs) and their contribution to tumor growth using mouse models harboring dysfunctional adipocytes. Specifically, we employ adipocyte-specific BECN1 KO (BaKO) mice, which exhibit lipodystrophy due to dysfunctional adipocytes. Our results reveal the activation of YAP/TAZ signaling in both CAAs and BECN1-deficient adipocytes, inducing adipocyte dedifferentiation and formation of a malignant TME. The additional deletion of YAP/TAZ from BaKO mice significantly restores the lipodystrophy and inflammatory phenotypes, leading to tumor regression. Furthermore, mice fed a high-fat diet (HFD) exhibit decreased BECN1 and increased YAP/TAZ expression in their adipose tissues. Treatment with the YAP/TAZ inhibitor, verteporfin, suppresses tumor progression in BaKO and HFD-fed mice, highlighting its efficacy against mice with metabolic dysregulation. Overall, our findings provide insights into the key mediators of CAA and their significance in developing a TME, thereby suggesting a viable approach targeting adipocyte homeostasis to suppress cancer growth.

Fig. 1. Dysfunctional adipocytes promote tumor progression in breast and colon cancer models.

a Tumor volumes and weights after subcutaneous injection of MC-38 into 6- week- old WT and adipocyte specific BECN1 KO (BaKO) mice (WT, n = 15; BaKO, n = 8). b Tumor volumes (WT, n = 18; BaKO, n = 12) and weights (WT, n = 8; BaKO, n = 10) after mammary fat pad injection of EO771 into 6-week-old WT and BaKO mice. c Average growth kinetics and incidence of breast tumors generated from 10 mammary glands of MMTV-PyMT BECN1 WT mice (PyWT, n = 16) and MMTV-PyMT BECN1 KO mice (PyBaKO, n = 16). d Average tumor mass isolated from each mammary gland and a representative image of the tumors from PyWT (n = 10) and PyBaKO (n = 6) mice. Mice were sacrificed at 23 weeks of age. e Metastasized tumor nodules counted from the lungs of PyWT (n = 13) and PyBaKO (n = 8) mice at 23 weeks of age. Representative image was taken from mice sacrificed at 23 weeks of age. f Kaplan–Meier plot of survival curve of PyWT and PyBaKO mice. Log-rank test p-value (p) and hazard ratio (HR) between PyWT (n = 13) and PyBaKO (n = 8) mice are indicated. g Schematic timeline of adipocyte-cancer co-injection. (4-OHT: 4-hydroxytamoxifen). h Western blotting analysis of protein lysates isolated from immortalized stromal vascular cells (imSVCs) before being mixed with cancer cells. i Tumor volumes and weights after subcutaneous injection of 4T1 cells mixed with WT or Becn1 KO adipocytes. Mice were sacrificed on the 39th day following co-injection (n = 3,4). Statistics were calculated using two-tailed unpaired Student’s t-test (a, b, d, i), ordinary two-way ANOVA (a, b, c, e, i), and Kaplan–Meier survival curve analysis (f). Data are shown as mean $\pm$SEM; *p$\le 0.05$, **p$\le 0.01$, ***p$\le 0.001$. Western blot results are representatives of at least three independent experiments.

Fig. 2. Dysfunctional adipocytes induce pro-inflammatory signals such as TNFɑ and LCN2, to support tumor growth.

a GSEA plot for RNA-seq result of BaKO iWAT. Significantly altered ‘Hallmark gene sets’ are aligned according to their normalized enrichment score (NES), p-val, and FDR. b Relative mRNA expression of TNFα downstream genes between WT and Becn1 KO imSVCs (n = 3, biologically independent samples per group). c Concentration of TNFα in the CM extracted from imSVCs of indicated genotypes using ELISA (n = 3, biologically independent samples per group). LPS (100 ng/mL) treatment for 24 h. d Cell viability assay performed on MC-38 (Normal n = 8; WT CM n = 4; KO CM n = 4, biologically independent cells per group) and EO771 e grown in normal or conditioned media extracted from WT or BECN1 KO adipocytes for 72 h (n = 3, biologically independent cells per group). f Mouse adipokine array performed in dot blot and heatmap analysis representing the altered adipokines. Samples were extracted from WT and BaKO mouse plasma (n = 2, each genotype). g Relative mRNA expression of adipokines associated with tumor growth in fully differentiated WT or BECN1 KO imSVCs (n = 3, biologically independent samples per group). h Western blot analysis of protein lysates isolated from imSVCs. i Relative mRNA expression of LCN2 in differentiated imSVCs treated with or without TNFα recombinant proteins for 24 h (n = 3, biologically independent samples per group). j Colony assay of EO771 co-cultured with WT or BECN1 KO adipocytes. A total of 50 cancer cells were seeded with or without lipocalin2 receptor siRNA (siLCN2R) treatment for 2 days before the co-culture (co-cultured adipocyte numbers are as indicated). Area (%) was quantified using ImageJ. This experiment was conducted once.Statistics were calculated using two-tailed unpaired Students t-test (b, c, d, e, g, i). Data are shown as mean $\pm$SEM; *p$\le 0.05$, **p $\le 0.01$, ***p$\le 0.001$. Western blot results are representatives of at least three independent experiments.

Fig. 3. Cancer-associated adipocytes exhibit similar phenotypes to BECN1-deficient adipocytes.

a Differential interference contrast microscopy image of adipocytes co-cultured with EO771 for the indicated duration. b Relative mRNA expression of adipogenic genes from differentiated 10T1/2 adipocytes co-cultured with EO771 breast cancer cells for 4 days (n = 3, biologically independent samples per group). c Western blotting analysis of protein lysates isolated from differentiated 10T1/2 adipocytes co-cultured with EO771 breast cancer cells over the time course. d Colony assay of 50 EO771 cells co-cultured with the indicated number of adipocytes. Each colony size was measured using imageJ (50 distinct colonies per group). This experiment was conducted once. e Image of naive and peri-tumoral iWAT samples resected from a mouse. f GSEA result of BaKO and naive adipose tissue ranked by NES. Gene sets that also appear to be differentially expressed in peritumoral WAT are colored in red and blue. g Venn diagram of GSEA results on BaKO and peritumoral WATs compared to naive adipose tissue; p$<0.01$, FDR < 0.05, NES < −1 or NES > 1 (h) GSEA plots for adipogenesis, (i) FA metabolism, and (j) YAP conserved signaling gene sets compared between naive, peritumoral, and BaKO WAT. Statistics were calculated using two-tailed unpaired students t-test (b, d). Data are shown as mean $\pm$SEM; *p $\le 0.05$, **p$\le 0.01$, ***p$\le 0.001$. Western blot results are representatives of at least three independent experiments.

Fig. 4. Autophagy inhibition in adipocytes is insufficient to induce YAP/TAZ signaling and tumor growth, while BECN1 regulates the Hippo pathway through interaction with Mob1.

a Western blot analysis of protein lysates isolated from differentiated 3T3L1 treated with autophagy inhibitors, Bafilomycin A (Baf) and Hydroxychloroquine (HCQ), for 48 h. b Representative IF image of HEK293T cells stained with YAP, TAZ, and DAPI after siRNA BECN1 and siATG7 transfection. Localization of YAP/TAZ was quantified using ImageJ. Refer to Supplementary Fig. 4d for the Western blot analysis. c Tumor volumes and weights measured after subcutaneous injection of MC-38 into 7-week-old WT and ATG7aKO (WT, n = 10; ATG7aKO, n = 12). d Tumor volumes and weights measured after mammary fat pad injection of EO771 into 7-week-old WT and ATG7aKO (WT, n = 10; ATG7aKO, n = 8). e Tumor volumes and weights measured after mammary fat pad injection of EO771 into 7-week-old mice treated with and without HCQ (50 mg/kg, injected everyday) (NT, n = 7; HCQ, n = 8). f Tumor volumes and weights measured after co-injection of EO771 and adipocytes. Adipocytes were pretreated with PBS or HCQ (40 μM) for 2 days. 2.5 × 10⁵ number of cancer cells and adipocytes were mixed to be subcutaneously injected into 7-week-old mice (Ctrl n = 7; HCQ n = 8). Statistics were calculated using ordinary two-way ANOVA (c, d) and two-tailed unpaired Students t-test (c, d, e, f). Western blot results are representatives of at least three independent experiments.

Fig. 5. BECN1-deficient adipocytes suppress adipogenesis while inducing YAP/TAZ signaling.

a Western blotting analysis of protein lysates isolated from differentiating adipocytes over the time course. b Representative IF images throughout 10T1/2 adipocyte differentiation. Image was taken from 2nd day to 8th day of differentiation. Adipocytes were stained with YAP/TAZ (red, upper panel) and β-catenin (red, lower panel), BODIPY (green), and DAPI (blue). c Western blotting analysis of protein lysates isolated from doxycycline-inducible Becn1 KO cell lines. Fully differentiated adipocytes with and without doxycycline (Doxy) treatment for the time indicated. d, e Representative IF images of mature adipocytes stained with YAP/TAZ and (e) β-catenin. Arrowheads indicate nuclear YAP/TAZ. f Representative IF images of PLA conducted on preadipocytes, adipocytes, and BECN1 KO adipocytes. Interaction between endogenous MOB1-LATS1 and LATS1-YAP1 were detected. g Representative IF image of PLA conducted on U2OS cells overexpressed with BECN1^GFP and MOB1^HA. h Western blotting analysis of protein lysates isolated from HEK293 cells overexpressed with BECN1^GFP, YAP1^FLAG, and MOB1^HA as indicated. The tag-antibodies were used to detect BECN1, YAP1, and MOB1. Endogenous p-YAP levels relative to YAP1 were quantified using imageJ. i Western blotting analysis of protein lysates isolated from imSVCs with and without 4-OHT treatment. j Western blotting analysis of protein lysates isolated from WT and BaKO inguinal white adipose tissues (iWATs). Mice were sacrificed at 8-weeks-old of age. Western blot results are representatives of at least three independent experiments.

Fig. 6. BaKO phenotypes are restored in BECN1/YAP/TAZ triple KO mice.

a Western blot analysis of protein lysates isolated from fully differentiated WT and adipocyte specific BECN1/YAP/TAZ KO (BYTaKO) SVCs. b–d Organ weights measured from 8-week-old WT, BaKO, and BYTaKO (WT, n = 16; BaKO, n = 10; BYTaKO, n = 10). e Heatmap analysis of TNFα downstream gene expression obtained from WT, BaKO, and BYTaKO iWAT. f Relative mRNA expression of BECN1, YAP, TAZ, and TNFα downstream genes confirmed by RT-qPCR (n = 2 mice per genotype). g Concentration of TNFα in WT, BaKO, BYTaKO mouse serum using ELISA (WT, n = 5; BaKO, n = 4; BYTaKO, n = 3). h Concentration of LCN2 in WT, BaKO, BYTaKO iWAT CM using ELISA. iWATs were resected and minced to obtain WAT CM (n = 3 mice per genotype). i Publicly available YAP1, TEAD1, and TEAD4 CHIP-seq reads around LCN2 gene loci. CHIP-seq was conducted in MCF10A and PC-9 cells. j Schematic representation of luciferase reporter construct containing LCN2 promoter region. Red stars indicate the TEAD binding motifs, and blue bar indicates the regions with high CHIP-seq reads. k Dual Luciferase assay conducted in HEK293 cells transfected with the luciferase reporter constructs (LCN2). After transfection, recombinant YAP1 protein was treated (1 μg, 48 h) (n = 3, biologically independent samples per group). l Colony assay of EO771 co-cultured with BECN1 or BECN1/YAP/TAZ KO adipocytes. A total of 30 cancer cells were seeded with differentiated adipocytes, with adipocyte numbers indicated. Sizes for each EO771 colony were quantified using ImageJ and presented relative to the control, expressed as log2-fold change (FC) (30 distinct colonies per group). This experiment was conducted once. Statistics were calculated using two-tailed unpaired Students t-test (b, c, d, g, h, l). Data are shown as mean $\pm$SEM; *p$\le 0.05$, **p$\le 0.01$, ***p$\le 0.001$. Western blot results are representatives of at least three independent experiments.

Fig. 7. Inhibition of YAP/TAZ activity suppresses the tumorigenic effect of BECN1-deficient adipocytes.

a Tumor volumes measured after MC-38 subcutaneously injected into 7-week-old WT, BaKO, and BYTaKO mice (WT n = 20; BaKO n = 12; BYTaKO n = 12). b Tumor volumes measured after EO771 subcutaneously injected into 7-week-old WT, BaKO, and BYTaKO (WT n = 16; BaKO n = 6; BYTaKO n = 8). c Heatmap analysis of adipogenesis and FA metabolism gene sets between WT, BaKO, and BYTaKO iWATs and their peritumoral WATs (labeled with red letter ‘T’) (n = 2 each). d Tumor volumes and weights after mammary fat pad injection of EO771 into 7- week-old mice treated with or without VP (30 mg/kg, injected every other day). VP treatment was initiated when tumors reached a volume of 150 mm³, and the mice were sacrificed on day 22 following injection (WT, n = 8; WT VP, n = 8; KO, n = 6; KO VP, n = 6). e Western blot analysis of protein lysates isolated from iWAT of Normal Chow Diet (NCD) and High Fat Diet (HFD)-fed mice. Mice were fed with HFD chow for 12 weeks. f Schematic timeline of NCD and HFD-fed mice treated with or without VP. g Tumor volumes and weights after mammary fat pad injection of EO771 into NCD or HFD-fed mice. Verteporfin(VP) treatment (30 mg/kg, injected every other day) was initiated when tumors reached a volume of 150 mm³, and the mice were sacrificed on day 26 following the injection (n = 12, each group). h Schematic illustration depicting the signaling mechanism identified in this study. Statistics were calculated using ordinary two-way ANOVA (a, b, d, g) and two-tailed unpaired students t-test (d, g). Data are shown as mean $\pm$SEM; *p$\le 0.05$, **p$\le 0.01$, ***p$\le 0.001$. Western blot results are representatives of at least three independent experiments.