Members of the Smad family of proteins are thought to play important roles in transforming growth factor ␤ (TGF-␤)-mediated signal transduction. In response to TGF-␤, specific Smads become inducibly phosphorylated, form heteromers with Smad4, and undergo nuclear accumulation. In addition, overexpression of specific Smad combinations can mimic the transcriptional effect of TGF-␤ on both the plasminogen activator inhibitor 1 (PAI-1) promoter and the reporter construct p3TP-Lux. Although these data suggest a role for Smads in regulating transcription, the precise nuclear function of these heteromeric Smad complexes remains largely unknown. Here we show that in Mv1Lu cells Smad3 and Smad4 form a TGF-␤-induced, phosphorylation-dependent, DNA binding complex that specifically recognizes a bipartite binding site within p3TP-Lux. Furthermore, we demonstrate that Smad4 itself is a DNA binding protein which recognizes the same sequence. Interestingly, mutations which eliminate the Smad DNA binding site do not interfere with either TGF-␤-dependent transcriptional activation or activation by Smad3/Smad4 cooverexpression. In contrast, mutation of adjacent AP1 sites within this context eliminates both TGF-␤-dependent transcriptional activation and activation in response to Smad3/Smad4 cooverexpression. Furthermore, concatemerized AP1 sites, in isolation, are activated by Smad3/Smad4 cooverexpression and, to a certain extent, by TGF-␤. Taken together, these data suggest that the Smad3/Smad4 complex has at least two separable nuclear functions: it forms a rapid, yet transient sequence-specific DNA binding complex, and it potentiates AP1-dependent transcriptional activation.

SUMMARY We present an integromic analysis of gene alterations that modulate transforming growth factor β (TGF-β)-Smad–mediated signaling in 9,125 tumor samples across 33 cancer types in The Cancer Genome Atlas (TCGA). Focusing on genes that encode mediators and regulators of TGF-β signaling, we found at least one genomic alteration (mutation, homozygous deletion, or amplification) in 39% of samples, with highest frequencies in gastrointestinal cancers. We identified mutation hotspots in genes that encode TGF-β ligands (BMP5), receptors (TGFBR2, AVCR2A, BMPR2), and Smads (SMAD2, SMAD4). Alterations in the TGF-β superfamily correlated positively with expression of metastasis-associated genes and with decreased survival. Correlation analyses showed the contributions of mutation, amplification, deletion, DNA methylation, and miRNA expression to transcriptional activity of TGF-β signaling in each cancer type. This study provides a broad molecular perspective relevant for future functional and therapeutic studies of the diverse cancer pathways mediated by the TGF-β superfamily.

The TGF-β pathway is a multifunctional signaling cascade with context-dependent roles in diverse biologic processes, including tumor promotion or suppression, metastasis, stem cell homeostasis, and immune suppression. Due to its highly context-dependent nature, decoding functional outcomes of the TGF-β pathway in specific tissues is highly challenging. Here, we present comprehensive genomic, transcriptomic and epigenomic analyses of the TGF-β pathway identified by 44 core pathway genes across 33 TCGA tumor types and 9125 samples. The core pathway genes involve TGF-β like ligands, receptors, intracellular SMAD molecules and adaptors. Although individual core pathway genes were rarely mutated or copy number altered in different cancer types, 41% of all samples have at least one genomic alteration in the TGF-β pathway, predominantly in the form of mutations. We identified a highly conserved TGF-β downstream gene expression signature associated with alterations in core pathway genes, suggesting that the alterations in the pathway have shared functional consequences. We observed a significant enrichment of the genomic alterations in gastrointestinal cancers (GI) with a distinct gene expression signature. The newly identified gene expression signature (over- or downregulation of key TGF-β downstream genes) in pan-cancer cohort was associated with significantly poor prognosis, particularly when it co-occurred with genomic alterations in the core pathway. Analysis of mutational hotspot sites revealed 6 genes with hotspots recurring in at least 9 (up to 78) mutational incidences. The hotspot mutations were also highly enriched in GI cancers. We identified previously characterized cancer mutation sites on SMAD4 and SMAD2 as hotspots mainly in GI cancers. We hypothesized novel functions to two of the newly identified hotpot sites through structural and trancriptomic analyses, and two other novel hotspot sites in the pathway await functional characterization. miRNA and epigenomic analyses revealed that TGF-β pathway activity is limited by epigenetic silencing or miRNA expression, especially in cancers with very low pathway gene expression levels. This multidimensional study provides the multifacefed landscape of TGF-β signaling in both individual disease and pan-cancer settings to guide future functional and therapeutic studies of this key cancer pathway. Citation Format: Anil Korkut, Sobia Zaidi, Rupa Kanchi, Ashton C. Berger, Gordon Robertson, Lawrence N. Kwong, Mike Datto, Jason Roszik, Shiyun Ling, Andre Schultz, Visweswaran Ravikumar, Ganiraju Manyam, Arvind Rao, Simon Shelley, Yuexin Liu, Zhenlin Ju, Donna Hansel, Guillermo de Velasco, Arjun Pennathur, Jesper B. Andersen, Colm J. O'Rourke, Kazufumi Ohshiro, Wilma Jogunoori, Nancy Gough, Shulin Li, Hatice Osmanbeyoglu, Andres Houseman, Shuyun Rao, Maciej Wiznerowicz, Jian Chen, Shoujun Gu, Wencai Ma, Jiexin Zhang, Pan Tong, Andrew D. Cherniack, Chuxia Deng, Linda Resar-Smith, Jaffer Ajani, The Cancer Genome Atlas Research Network, John N. Weinstein, Lopa Mishra, Rehan Akbani. A pan-cancer atlas of genomic, epigenomic and transcriptomic alterations in the TGF-β pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3413.

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