Association of subcellular localization of TEAD transcription factors with outcome and progression in pancreatic ductal adenocarcinoma
Richard Drexler 1, Rebecca Fahy 2, Mirco Küchler 2, Kim C Wagner 2, Tim Reese 2, Mareike Ehmke 3, Bernd Feyerabend 4, Moritz Kleine 5, Karl J Oldhafer 2
Abstract
Background
Transcriptional enhanced associated domain (TEAD) transcription factors are nuclear effectors of several oncogenic signalling pathways including Hippo, WNT, TGF-ß and EGFR pathways that interact with various cancer genes. The subcellular localization of TEAD regulates the functional output of these pathways affecting tumour progression and patient outcome. However, the impact of the TEAD family on pancreatic ductal adenocarcinoma (PDAC) and its clinical progression remain elusive.
Methods
A cohort of 81 PDAC patients who had undergone surgery was established. Cytoplasmic and nuclear localization of TEAD1, TEAD2, TEAD3 and TEAD4 was evaluated with the immunoreactive score (IRS) by immunohistochemistry (IHC) using paraffin-embedded tissue. Results were correlated with clinicopathological data, disease-free and overall survival.
Results
Nuclear staining of all four TEADs was increased in pancreatic cancer tissue. Patients suffering from metastatic disease at time of surgery showed a strong nuclear staining of TEAD2 and TEAD3 (p < 0.05). Furthermore, a nuclear > cytoplasmic ratio of TEAD2 and TEAD3 was associated with a shorter overall survival and TEAD2 emerged as an independent prognostic factor for disease-free survival.
Conclusion
Our study underlines the importance of TEAD transcription factors in PDAC as a nuclear localization was found to be associated with metastatic disease and an unfavourable prognosis after surgical resection.
Introduction
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and remains a challenging disease with a poor prognosis. The 5-year survival rate is between 4 and 8%, with surgical resection remaining the only curative option [1]. At the time of diagnosis only 15–20% of patients are eligible for surgery and up to 50% of patients display hepatic metastasis [2]. Ultimately 70% of patients die from metastatic disease [3].
The transcriptional enhanced associated domain (TEAD) family consists of four highly conserved TEAD/TEF transcription factors (TEAD 1, TEAD 2, TEAD 3 and TEAD 4). The TEA binding domain is common to all four and transcriptional coactivators are necessary for transcription activation [4]. TEADs integrate with and regulate many signal transduction pathways [5]. It has been proven that TEADs are the central transcription factors of the Hippo pathway with most studies from the last few years focusing on the interaction between Yes-associated protein (YAP) and TEAD [[6], [7], [8]].
Hippo independent regulators and modulators have only recently come to light. However, TEAD activity is also influenced by upstream signal transduction pathways, such as Wnt, TGF-ß and epidermal growth factor pathways [5]. Furthermore, the discovery of TEADs molecular mechanisms, such as post-translational modifications and nucleocytoplasmic shuttling, have highlighted the importance of TEAD’s transcriptional activity [[9], [10], [11], [12]]. It is indispensable that TEAD is located in the cell nucleus in order for interaction with various cofactors and to allow for its full transcriptional activity. There is a broad spectrum of stressors like osmotic stress, high cell density and cell suspension, inducing TEAD cytoplasmic translocation [12]. However, other cellular events like energy starvation, oxidative stress or cytotoxic agents lead to the inhibition of TEAD transcriptional outputs, but do not alter TEADs’ subcellular location or level of expression [5]. One underlying mechanism is a p38 MAPK-induced cytoplasmic translocation of TEAD in a Hippo-independent manner, indicating that influencing the nucleocytoplasmic shuttling of TEAD could be an effective therapeutic target [12].
As the functional output of several interacting pathways, TEAD plays a major role during organ development, cell growth, regeneration and tissue homeostasis. This is achieved through their transcriptional target genes, such as connective tissue growth factor (CTGF), Cysteine-rich angiogenic inducer (Cyr61) and Myc. TEAD hyperactivity and overexpression has been observed in multiple stages of cancer progression and it is well known that TEAD contributes to tumour initiation, promotes cancer progression, epithelial-mesenchymal transition (EMT) and metastasis [5]. Overexpression of TEAD can be seen in various malignancies, such as medulloblastoma, lung, ovarian and breast cancer [[13], [14], [15], [16], [17]].
Additional data have shown that the activation of TEAD target genes mediate metastases in various malignancies. In breast cancer the activation of CTGF enhances the colonisation of malignant cells and myocardin-related transcription factor (MRTF) induced breast cancer cell metastases to the lung [18,19]. In colorectal cancer TEAD activation is triggered by RAR-γ via the Hippo pathway, which in turn promotes EMT, invasion and metastasis [20]. Nonetheless higher TEAD expression and its localization in the nucleus of colorectal cancer cells induced EMT and metastasis via a Hippo-independent mechanism [21]. In bone metastasis the activation of TEAD induces ROR1-HER3-mediated osteoclast differentiation through the Hippo-YAP pathway [22].
Interestingly in PDAC, a few studies revealed that disrupting the YAP-TEAD interaction could be a promising therapeutic target to supress tumour cell growth and metastatic processes [[23], [24], [25]]. In addition, Cebola et al. unveiled a central role of TEAD and YAP as regulators of multipotent pancreatic progenitors [26]. Rozengurt et al. found that numerous TEAD target genes have been associated with an unfavourable prognosis [27]. However, no study investigated the prognostic impact of TEAD staining itself in PDAC.
The aim of our study was to demonstrate the clinical impact of TEADs in patients with PDAC, regardless of their upstream signalling pathways.
Section snippets
Ethics approval
All patients’ data were fully anonymised, and the study was performed according to the standards set in the Declaration of Helsinki 1975. The tumour tissue used was remaining from material that initially had been collected for diagnostic purposes. All diagnostic procedures had already been fully completed when the samples were retrieved for the study. The study was approved by the Ethics Committee Hamburg, Germany (approval number PV5510).
Patients characteristics
A total of 81 patients (female, n = 41; male, n = 40.
Study population
A total of 81 patients who had been diagnosed with PDAC and undergone surgery between 2012 and 2018 were enrolled in this study. The patients had a median age of 65.2 years and 41 were female (50.6%). 6 patients (7.4%) received neoadjuvant chemotherapy with mFOLFIRINOX. The majority of the tumours was located in the pancreas head (80.2%). Due to the most common tumour location, a pancreaticoduodenectomy (PDPP) was performed in the most cases (71.6%).
Discussion
In our study, we used immunohistochemical techniques to assess protein levels and subcellular localization of all members of the TEAD family. This was carried out on 81 patients diagnosed with PDAC. As TEAD could only interact with their target genes when located in the cell nucleus, we wondered if a nuclear localization of TEAD is associated with tumour progression and an unfavourable prognosis following surgery.
Conclusion
Our study demonstrated that a nuclear shift of TEAD2 and TEAD3 could have a major impact on metastatic and progressive disease in PDAC. Furthermore, TEAD2 has emerged as an independent prognostic factor for OS and DFS and emphasizes the clinical relevance of the TEAD family. According to our results, inhibiting TEAD proteins directly or influencing the MYF-01-37 nucleocytoplasmic shuttling towards a cytoplasmic sequestration of TEADs could be a novel and very promising therapeutic approach.
Declaration of competing interest
No potential conflicts of interest were disclosed by the authors.
Acknowledgements
This work is part of the M.D. thesis of R. Drexler and the diploma thesis of R. Fahy.