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A Novel Regulatory Network of ∆NP63-TGFβ-MicroRNAs Regulates Cancer Metastasis
By Sahana Shankar, Ph.D. Candidate
P53 is a well-known and widely-studied tumor suppressor gene that regulates cell division and apoptosis. P53 mutations are a hallmark of many cancers and an attractive drug target. The regulation of p53 involves various transcription factors and signaling pathways. A recent study in Cancer Research focused on the transcription factor ∆Np63 to understand how it controls the p53 and TGFβ pathways during cancer progression.
∆Np63 is one of the isoforms of the TP63 gene, which lacks the full-length transactivation domain. It is expressed in the epidermis and is essential for the terminal differentiation of epidermal cells. Previous work has shown that ∆Np63 is overexpressed in many primary tumors. It is found to be downregulated in tumor cells with high metastatic capacity. Interestingly, metastases have a high expression of ∆Np63, suggesting that it is under a strict spatiotemporal regulation, which is important for tumor cells to move from the primary tumor to distant organs.
Using human breast cancer cell lines, breast cancer metastatic models in mice and human samples, and biopsies, Dr. Elsa Flores’ lab at the University of South Florida studied the oscillatory expression of ∆Np63 and its role in tumor invasion and progression. ∆Np63 was found to be downregulated in invasive breast cancer, indicating that it must be suppressed for metastasis. Loss of ∆Np63 correlated with the advancement of primary tumors.
∆Np63 is Required for Migratory and Invasive Behaviour of Breast Cancer Cells
The depletion of ∆Np63 in various breast cancer cell lines showed an increased expression of EMT-associated factors that enable the epithelial to mesenchymal transition required for metastasis. Using an inducible ∆Np63, the team studied its effect on different stages of the metastatic cascade. Nude mice with primary breast cancer were divided into three groups- group 1 with high ∆Np63 expression, group 2 with oscillatory ∆Np63 expression (low and high), and group 3 with low ∆Np63 expression.
While group 2 showed the highest metastasis to lungs, group 3 showed lowered expression of Ki67 indicating reduced tumor cell proliferation. Besides, the metastatic frequency was higher in group 3 when compared to group 1. Strikingly, ∆Np63 depletion inhibited cell proliferation but also triggered cell migration and invasion. Taken together, these data suggest that oscillatory expression of ∆Np63 in tumor cells favors metastasis.
To study metastatic colonization, the investigators used a similar inducible ∆Np63 in human breast cancer cell line MCF-10A labeled with a red fluorescent protein and introduced these tumor cells into nude mice via tail vein injection and quantified their migration into lungs by flow cytometry. Group 1 and group 2 had a higher percentage of lung colonization as compared to group 3. In continuation, mice injected with ∆Np63 were aged for 8 weeks to study lung metastases. Group 2 turned out to have the highest number of tumor nodules and metastatic lesions in the lungs, indicating that ∆Np63 is required for extravasation and colonization of secondary tissues.
TGFβ/microRNAs/ΔNp63 Axis
In an attempt to find upstream regulators, the authors zeroed in on TGFβ, which shares the oscillatory behavior of ∆Np63 and is implicated in many cancers. ∆Np63 was downregulated upon the addition of TGFβ in human breast cancer cell lines- MCF10A, MCF1-DCIS, and MCF10CA1D. Inhibition of TGFβ signaling or the knockdown of Smad3 (downstream regulator of TGF-β) restored the expression of ∆Np63. Conversely, the overexpression of exogenous ∆Np63 counteracted the enhanced migration and invasion due to exogenous TGFβ. Inhibition of endogenous TGF-β also resulted in increased ∆Np63 levels, indicating that ∆Np63 is a part of the Smad2/3-dependent TGFβ signaling pathway.
To understand the regulation of ∆Np63 by TGFβ, the authors screened microRNAs upregulated in MCF cell lines upon TGFβ treatment and found four TGFβ-regulated microRNAs- miR-22-3p, miR-30a-5p, miR-203a-3p and miR-222-3p, which control ∆Np63 expression in breast cancer. Further analyses revealed that these four are direct targets of Smad-dependent TGFβ signaling and are required for ∆Np63 silencing. Their inhibition reduced migration and invasion capacity of breast cancer cell lines, indicating a possible feedback loop among the four miRNAs and TGFβ to modulate ∆Np63 levels.
The authors finally confirmed the effects of the TGFβ/microRNAs/ΔNp63 axis in the mouse model and a wide panel of human cancer cell lines by injecting TGFβ and monitoring microRNAs and ∆Np63 expression. Bioinformatic analyses of the TGFβ and ΔNp63 signatures in many cancer types showed that the ΔNp63 regulation by TGFβ is more relevant in breast cancers with low levels of ΔNp63.
In this comprehensive study of ΔNp63, the authors establish that it promotes breast primary tumor development, extravasation, and an oscillatory expression of ΔNp63 is essential for metastasis. It is spatiotemporally regulated by TGFβ and associated microRNAs and is a promising drug target for breast cancer.
Editor: Rajaneesh K. Gopinath, Ph.D.
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References
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