Biochemical and Biophysical Research Communications
Wwox suppresses breast cancer cell growth through modulation of the hedgehog–GLI1 signaling pathway
Introduction
Breast cancers are complex diseases that are caused by alterations of both genetic and epigenetic factors [1], [2]. Despite advances in chemotherapy, radiotherapy, and adjuvant hormonal therapy, one third of patients with breast cancers relapse and die of disease. New therapeutic strategies are needed to improve this outcome.
The tumor-suppressor gene, Wwox (WW domain containing oxidoreductase), spans one of the most active fragile sites, FRA16D, at chromosome 16q23.3-24.1, a region exhibiting loss of heterozygosity in breast, prostate, and other cancers [3], [4]. Wwox encodes a 414 amino acid, 46 kDa protein. The full-length Wwox possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence (NLS), and a C-terminal short-chain alcohol dehydrogenase/reductase (SDR) domain [5]. The WW domains are believed to be involved in protein–protein interactions, including a number of transcription factors [6]. The SDR region of Wwox may be involved in sex-steroid metabolism due to its high amino-acid sequence homology to specific oxidoreductases [7].
Numerous studies showed Expression of Wwox is either altered or lost from epigenetic modification in multiple malignant cancers, such as breast, esophageal, lung, ovarian, colon, prostate, and gastric carcinomas [4], [8], [9], [10]. Restoration of Wwox gene prevents the growth of lung cancer [11], prostate cancer [12], and breast cancer [13]. loss of one allele increased the incidence of mammary tumor formation [14], thus confirming that Wwox is a tumor suppressor. Previous works showed that the Wwox physically and functionally interacts with PPxY-containing proteins such as p73, AP-2gamma, c-Jun, RUNX2, ErbB4, TMEM207 and DeltaNp63alpha via its WW1 domain [6], [15], [16], [17], [18], [19], [20].
The Hedgehog (Hh) family of proteins regulates a wide variety of developmental processes, and malfunction of this pathway has been linked to numerous human disorders including cancer [21], [22]. In vertebrates the Hh pathway begins with the binding of Hh ligands to the Patched receptors on the membrane, which negatively regulates the 7-transmembrane protein Smoothened (Smo), which regulate the transcriptional activity of Gli1 zinc-finger transcription factor [23]. A recent study demonstrated that Gli1 composed of a combination of two PPxYs and a phospho-serine/proline(pSP) motifs in C-terminal region. Numb recruits Gli1 into the complex with Itch to target Gli1 protein for ubiquitination and degradation [24].
In this work, we describe a physical and functional interaction between the Wwox tumor suppressor and the Gli1 zinc-finger transcription factor. We show that Wwox binds Gli1, changes its cellular localization, inhibits its expression in vitro, and counteracts Gli1-mediated effects to breast cancer cell growth.
Section snippets
Cell culture
Breast cancer-derived cell lines were a gift from Dr. Shao Zhi-min (Fudan University, Shanghai, China). The human cancer cell lines MCF-7, T47D, SK-BR-3and ZR-75-30 were maintained in DMEM medium plus 10% fetal bovine serum (Gibco) and cultured in 5% CO2 humidified atmosphere. MDA-MB-231 were maintained in L15 medium plus 10% FBS and cultured in 100% air humidified atmosphere.
Plasmids
Human Gli1 expression vector pRK5-Gli1 and pRK5-Myc-Sufu was kindly provided by Dr. Steven Y. Cheng (Nanjing Medical
Expression of Wwox negatively correlates with Gli1 expression in breast cancer cells
To study the effects of restoration of Wwox on Gli1 expression in breast cancer cells, we first examined the endogenous mRNA expression of Wwox, Gli1 and ER in several human breast cancer cell lines by RT-PCR (Fig. 1A). In accordance with previous reports [32], [33], [34], [35], our result showed that ER mRNA was detectable in estrogen-dependent cell lines, including MCF-7 and T47D cells, but not detectable in estrogen-independent cell lines, such as MDA-MB-231, SK-BR-3 and ZR-75-30 cells. The
Discussion
In the current study, we first demonstrate that Wwox down-regulates Gli1 in breast cancer cells. Several groups analyzed Wwox and Gli1 expression in breast cancer in cell lines, but their results are not entirely consistent. For example, Mukherjee showed high Gli1 mRNA levels in estrogen-dependent cells (MCF-7, T47D) and estrogen-independent cells (MDA-MB-231 and SK-BR-3) [29]. and Zhao J. and Zhang X. found lacking of Gli1 mRNA expression in MCF-7 cells and high levels in T47D, MDA-MB-231,
Acknowledgments
This work was supported by the National Science Foundation of China (81072175; 81372854; 81102010) and the Shanghai Committee of Science and Technology, China (Grant No. 13NM1401504).
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WW45 inhibits breast cancer cell proliferation by the Hedgehog signaling pathway
2021, American Journal of Translational ResearchThe WWOX gene in brain development and pathology
2020, Experimental Biology and MedicineThe WWOX Gene Influences Cellular Pathways in the Neuronal Differentiation of Human Neural Progenitor Cells
2019, Frontiers in Cellular NeuroscienceWWOX tumor suppressor gene in breast cancer, a historical perspective and future directions
2018, Frontiers in Oncology
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Anwen Xiong and Li Wei contributed equally to this work, and all should be considered first author.