Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/β-catenin signaling are frequently observed, the β-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/β-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/β-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/β-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/β-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/β-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/β-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/β-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this β-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/β-catenin inhibitor. These results show promise for the further clinical development of Wnt/β-catenin inhibitors in ACC and unveil a novel Wnt/β-catenin-regulated transcriptome.

Adrenocortical carcinoma (ACC) is a rare, but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. Improved understanding of transcriptional programs engaged in ACC will help direct rational, targeted therapies. While activating mutations in Wnt/β-catenin signaling are frequently observed, the β-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we used independent component analysis and identified a novel Wnt/β-catenin-associated signature in ACC predictive of poor survival. This signature was enriched for the extracellular matrix (ECM), suggesting a potential role for Wnt/β-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression strongly correlated with both Wnt/β-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/β-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced expression of COL11A1 and other ECM components, and decreased viability of cancer cells in vitro. To investigate the preclinical potential of Wnt/β-catenin inhibition in vivo, we developed and characterized a novel orthotopic xenograft model utilizing minimally invasive techniques. Treatment with the newly developed Wnt/β-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth in this preclinical model. Together, our data supports that inhibition of aberrantly active Wnt/β-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this β-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/β-catenin inhibitor. These results show promise for further clinical development of Wnt/β-catenin inhibitors in ACC and unveil a novel Wnt/β-catenin-regulated transcriptome.

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Adrenocortical carcinoma (ACC) is an aggressive cancer that affects 1-2 people per million in the United States annually. The only option for cure is surgical resection, and the 5-year survival rate for ACC remains low at 35%. Improved understanding of this disease is needed to develop rational therapies. Genomic alterations activating Wnt/Beta-catenin signaling occur in approximately 40% of ACCs and are associated with poor prognosis. However, the biologic consequences of constitutive Wnt/Beta-catenin activation in ACC are poorly understood. Treatment with Beta-catenin inhibitors BC2059 and PKF115-584 decreases viability in the NCI-H295R ACC cell line, which harbors an activating CTNNB1 (Beta-catenin) mutation. In addition, BC2059 significantly inhibits NCI-H295R tumor growth in an orthotopic xenograft model. Our data supports the hypothesis that Wnt/Beta-catenin regulates ACC cell growth and survival. To better characterize transcriptional programs engaged by Wnt/Beta-catenin signaling in ACC, we performed independent component analysis on The Cancer Genome Atlas (TCGA) ACC transcriptome dataset to identify components of coordinately expressed genes. One component is significantly enriched for Wnt signaling activity and is strongly associated with somatic CTNNB1 (Beta-catenin) mutation. The Wnt-enriched component is also enriched for extracellular matrix (ECM)-receptor interaction, including (but not restricted to) expression of COL11A1 (Collagen alpha-1(XI)), COL26A1 (Collagen alpha-1(XXVI)), LAMC3 (Laminin, gamma 3), and ITGA2 (Integrin, alpha 2), suggesting that Wnt/Beta-catenin is regulating composition of the ACC microenvironment. Current literature supports that ACC has relatively low contribution from stromal cells, suggesting that tumor-cell-derived ECM may have substantial biologic effect in the tumor microenvironment. To follow up on these observations, we performed immunohistochemical staining on tissue microarrays containing 97 ACC samples. We observed a strong correlation of COL11A1 expression and nuclear Beta-catenin localization, suggesting COL11A1 may be regulated by Wnt/Beta-catenin. Furthermore, COL11A1 expression is significantly associated with decreased overall survival and decreased event-free survival. Cell culture studies confirm that expression of COL11A1, COL26A1, and LAMC3 is significantly reduced in NCI-H295R cells treated with PKF115-584 or BC2059, consistent with our hypothesis that Wnt/Beta-catenin regulates expression of ECM components in ACC. These results illustrate a novel role for Wnt/Beta-catenin activity in ACC, and suggest that Beta-catenin-associated expression of ECM components may contribute to cancer cell survival and disease progression. Citation Format: Morgan K. Penny, Antonio M. Lerario, Chris LaPensee, Thomas J. Giordano, Ruolan Han, Erika A. Newman, Gary D. Hammer. The role of the Wnt/Beta-catenin pathway in adrenocortical carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3955.

Adrenocortical carcinoma (ACC) is a rare and often aggressive cancer that affects 1-2 people per million in the United States annually. Genomic alterations activating canonical Wnt signaling occur in approximately 40% of ACCs and are associated with poor prognosis. However, the biological consequences of constitutive canonical Wnt activation in ACC are poorly understood. To better characterize the transcriptional programs that are engaged in ACC, we performed independent component analysis on The Cancer Genome Atlas (TCGA) ACC transcriptome dataset to identify components of coordinately expressed genes. One of the components identified was significantly enriched for Wnt signaling (p = 0.001) and was strongly associated with somatic CTNNB1 (β-catenin) mutations (p = 2.276e-07). Interestingly, this Wnt-enriched component also showed enrichment for extracellular matrix (ECM)-receptor interaction (p = 0.00139), including (but not restricted to) expression of COL11A1 (Collagen alpha-1(XI)), LAMC3 (Laminin, gamma 3), and ITGA2 (Integrin, alpha 2), suggesting that Wnt signaling is regulating cell-ECM interactions and ECM composition in ACC. To follow up on this observation, we performed immunohistochemical staining on tissue microarrays (TMAs) containing 97 ACC samples. We observed a strong correlation of COL11A1 expression and nuclear β-catenin localization (p = 0.0088), suggesting COL11A1 expression may be regulated by canonical Wnt signaling. Furthermore, COL11A1 expression was associated with decreased overall survival (p = 0.0003) and decreased event-free survival (p = 0.0075). To determine whether ECM and ECM-receptor genes are regulated by canonical Wnt activity, the NCI-H295R human ACC cell line, harboring an activating CTNNB1 mutation, was used. Cells were treated with PKF115-584, an inhibitor of canonical Wnt signaling that disrupts β-catenin interaction with TCF/LEF transcription factors. PKF115-584 treatment significantly reduced expression of COL11A1, LAMC3, and ITGA2 in NCI-H295R cells at early timepoints (p≤0.05), consistent with our hypothesis that canonical Wnt signaling regulates expression of ECM components in ACC. At late timepoints following PKF115-584 treatment, NCI-H295R cell viability decreased, indicating that canonical Wnt activity may regulate cell survival in ACC. These results illustrate a novel role for canonical Wnt activity in ACC, and suggest that β-catenin-regulated transcription of ECM and ECM-receptor components may promote cancer cell survival and aggressive disease. Future studies are aimed at characterizing the contribution of extracellular proteins to ACC phenotypes. Citation Format: Morgan K. Penny, Antonio M. Lerario, Chris LaPensee, Thomas J. Giordano, Gary D. Hammer. Canonical Wnt-associated extracellular matrix in adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1894.