Hippo/MST blocks breast cancer by downregulating WBP2 oncogene expression via miRNA processor Dicer.

WBP2 transcription coactivator is an emerging oncoprotein and a key node of convergence between EGF and Wnt signaling pathways. Understanding how WBP2 is regulated has important implications for cancer therapy. WBP2 is tightly controlled by post-translational modifications, including phosphorylation and ubiquitination, leading to changes in subcellular localization, protein-protein interactions, and protein turnover. As the function of WBP2 is intricately linked to YAP and TAZ, we hypothesize that WBP2 is negatively regulated by the Hippo tumor suppressor pathway. Indeed, MST is demonstrated to negatively regulate WBP2 expression in a kinase-dependent but LATS-independent manner. This was observed in the majority of the breast cancer cell lines tested. The effect of MST was enhanced by SAV and concomitant with the inhibition of the transcription co-activation, in vitro and in vivo tumorigenesis activities of WBP2, resulting in good prognosis in xenografts. Downregulation of WBP2 by MST involved miRNA but not proteasomal or lysosomal degradation. Our data support the existence of a novel MST-Dicer signaling axis, which in turn regulates both WBP2 CDS- and UTR-targeting miRNAs expression, including miR-23a. MiR-23a targets the 3'UTR of WBP2 mRNA directly. Significant inverse relationships between WBP2 and MST or miR23a expression levels in clinical specimens were observed. In conclusion, WBP2 is a target of the Hippo/MST kinase; MST is identified as yet another rheostat in the regulation of WBP2 and its oncogenic function. The findings have implications in targeted therapeutics and precision medicine for breast cancer.

Wnt Signaling Promotes Breast Cancer by Blocking ITCH-Mediated Degradation of YAP/TAZ Transcriptional Coactivator WBP2.

Cross-talk between the Hippo and Wnt pathways has been implicated recently in breast cancer development, but key intersections have yet to be fully defined. Here we report that WBP2, a transcription coactivator that binds the Hippo pathway transcription factor YAP/TAZ, contributes to Wnt signaling and breast cancer pathogenesis. Clinically, overexpression of WBP2 in breast cancer specimens correlated with malignant progression and poor patient survival. In breast cancer cells, nuclear entry and interaction of WBP2 with ß-catenin was stimulated by Wnt3A, thereby activating TCF-mediated transcription and driving malignant invasive character. Mechanistic investigations showed WBP2 levels were controlled by the E3 ligase ITCH, which bound and target WBP2 for ubiquitin-dependent proteasomal degradation. Accordingly, ITCH silencing could elevate WBP2 levels. Wnt signaling upregulated WBP2 by disrupting ITCH-WBP2 interactions via EGFR-mediated tyrosine phosphorylation of WBP2 and TAZ/YAP competitive binding. Conversely, ITCH-mediated downregulation of WBP2 inhibited TCF/ß-catenin transcription, in vitro transformation, and in vivo tumorigenesis. We identified somatic mutations in ITCH, which impaired its ability to degrade WBP2 and to block its function in cancer, even while retaining binding capacity to WBP2. Thus, the Wnt pathway appeared to engage WBP2 primarily by affecting its protein stability. Our findings show how WBP2/ITCH signaling functions to link the intricate Wnt and Hippo signaling networks in breast cancer. Cancer Res; 76(21); 6278-89. ©2016 AACR.

Mitf regulates osteoclastogenesis by modulating NFATc1 activity.

Transcription factors Mitf and NFATc1 share many downstream targets that are critical for osteoclastogenesis. Since RANKL signals induce/activate both NFATc1 and Mitf isoform-E (Mitf-E), a tissue-restricted Mitf isoform in osteoclasts, it is plausible that the two factors work together to promote osteoclastogenesis. Although Mitf was shown to function upstream of NFATc1 previously, this study showed that expression of Mitf had little effects on NFATc1 and NFATc1 was critical for the induction of Mitf-E. In Mitf(mi/mi) mice, the semi-dominant mutation in Mitf gene leads to arrest of osteoclastogenesis in the early stages. However, when stimulated by RANKL, the Mitf(mi/mi) preosteoclasts responded with a significant induction of NFATc1, despite that the cells cannot differentiate into functional osteoclasts. In the absence of RANKL stimulation, very high levels of NFATc1 are required to drive osteoclast development. Our data indicate that Mitf functions downstream of NFATc1 in the RANKL pathway, and it plays an important role in amplifying NFATc1-dependent osteoclastogenic signals, which contributes to the significant synergy between the two factors during osteoclastogenesis. We propose that Mitf-E functions as a tissue-specific modulator for events downstream of NFATc1 activation during osteoclastogenesis.

CXCL3 is a potential target for breast cancer metastasis.

Secreted proteins are an attractive minefield for cancer drug targets. An iTRAQ-based tandem mass spectrometry approach was employed to relatively quantify proteins in the secretomes of four isogenic breast cancer cell lines with increasing metastatic potential. CXCL3 was found to be upregulated in aggressive cancer cells. SiRNA and antibody neutralization studies supported a role of CXCL3 in metastatic processes. Meta-analysis of the mRNA level of CXCL3 in 1881 breast tumors supported a role of CXCL3 in clinical breast cancer. Our results support a functional role of CXCL3 in breast cancer metastasis and as a viable target for cancer therapy.

Subcellular localization of Mitf in monocytic cells.

Microphthalmia-associated transcription factor (Mitf) is a transcription factor that plays an important role in regulating the development of several cell lineages. The subcellular localization of Mitf is dynamic and is associated with its transcription activity. In this study, we examined factors that affect its subcellular localization in cells derived from the monocytic lineage since Mitf is present abundantly in these cells. We identified a domain encoded by Mitf exon 1B1b to be important for Mitf to commute between the cytoplasm and the nucleus. Deletion of this domain disrupts the shuttling of Mitf to the cytoplasm and results in its retention in the nucleus. M-CSF and RANKL both induce nuclear translocation of Mitf. We showed that Mitf nuclear transport is greatly influenced by ratio of M-CSF/Mitf protein expression. In addition, cell attachment to a solid surface also is needed for the nuclear transport of Mitf.

Mitf induction by RANKL is critical for osteoclastogenesis.

Microphthalmia-associated transcription factor (Mitf) regulates the development and function of several cell lineages, including osteoclasts. In this report, we identified a novel mechanism by which RANKL regulates osteoclastogenesis via induction of Mitf isoform E (Mitf-E). Both Mitf-A and Mitf-E are abundantly present in osteoclasts. Unlike Mitf-A, which is ubiquitously expressed and is present in similar amounts in macrophages and osteoclasts, Mitf-E is almost nondetectable in macrophages, but its expression is significantly up-regulated during osteoclastogenesis. In addition to their different expression profiles, the two isoforms are drastically different in their abilities to support osteoclastogenesis, despite sharing all known functional domains. Unlike Mitf-A, small amounts of Mitf-E are present in nuclear lysates unless chromatin is digested/sheared during the extraction. Based on these data, we propose a model in which Mitf-E is induced during osteoclastogenesis and is closely associated with chromatin to facilitate its interaction with target promoters; therefore, Mitf-E has a stronger osteoclastogenic activity. Mitf-A is a weaker osteoclastogenic factor, but activated Mitf-A alone is not sufficient to fully support osteoclastogenesis. Therefore, this receptor activator for nuclear factor-kappaB ligand (RANKL)-induced Mitf phenomenon seems to play an important role during osteoclastogenesis. Although the current theory indicates that Mitf and its binding partner Tfe3 are completely redundant in osteoclasts, using RNA interference, we demonstrated that Mitf has a distinct role from Tfe3. This study provides the first evidence that RANKL-induced Mitf is critical for osteoclastogenesis and Mitf is not completely redundant with Tfe3.

Ripe areca nut extract induces G1 phase arrests and senescence-associated phenotypes in normal human oral keratinocyte.

Around 200-600 million Asians chew areca (also called betel), which contains a mixture of areca nut and other ingredients. Epidemiological evidences indicated that areca use is tightly linked to oral carcinogenesis. This study investigated the effects of ripe areca nut extract (ANE) on cultured normal human oral keratinocyte (NHOK). Acute subtoxic ANE treatment inhibited DNA synthesis and induced cell cycle arrest at G1 phase in early passage (< 4th passage) cells. This was accompanied by a slight increase in the sub-G1 cellular fraction. O6-Methylguanine-DNA methyltransferase (MGMT), Hsp27 and p38MAPK was upregulated. p16 and p21 were remarkably upregulated early and declined afterwards. In contrast, the increase of dephosphorylated Rb seemed to be secondary to the episodes of p16 and p21 upregulation. To simulate the chronic areca exposure in vivo, constant ANE treatment in serial NHOK culture was performed. It resulted in a significant decrease in the population doubling, increase in senescence-associated beta-galactosidase (SA-beta-Gal) and decrease in cell proliferation in NHOK of late passages (> or = 4th passage). Induction of senescence-associated phenotypes, G2/M accumulation and genomic instability following long-term ANE treatment were also observed in a low-grade oral carcinoma cell. ANE-treated NHOK also had a higher nuclear factor-kappaB (NF-kappaB) fraction and a lower cytosolic IkappaBalpha level relative to the control in late passages. Moreover, electrophoretic mobility shift assay (EMSA) indicated that ANE treatment shifted the NF-kappaB complex from high mobility position to lower mobility position in late-passaged NHOK. ANE treatment also upregulated IL-6 and cyclooxygenase-2 (COX-2) mRNA expressions in late-passaged NHOK. In summary, our findings suggest that ANE induces the cell cycle arrest at G1/S phase and the occurrence of senescence-associated phenotypes of NHOK. The upregulation of p38MAPK, p16, p21, NF-kappaB, IL-6 and COX-2 are likely to participate in the control of these impacts.