Qinong Ye

Hepatitis B virus X protein represses miRNA-148a to enhance tumorigenesis

MicroRNAs (miRNAs) have been shown to be dysregulated in virus-related cancers; however, miRNA regulation of virus-related cancer development and progression remains poorly understood. Here, we report that miR-148a is repressed by hepatitis B virus (HBV) X protein (HBx) to promote cancer growth and metastasis in a mouse model of hepatocellular carcinoma (HCC). Hematopoietic pre-B cell leukemia transcription factor-interacting protein (HPIP) is an important regulator of cancer cell growth. We used miRNA target prediction programs to identify miR-148a as a regulator of HPIP. Expression of miR-148a in hepatoma cells reduced HPIP expression, leading to repression of AKT and ERK and subsequent inhibition of mTOR through the AKT/ERK/FOXO4/ATF5 pathway. HBx has been shown to play a critical role in the molecular pathogenesis of HBV-related HCC. We found that HBx suppressed p53-mediated activation of miR-148a. Moreover, expression of miR-148a was downregulated in patients with HBV-related liver cancer and negatively correlated with HPIP, which was upregulated in patients with liver cancer. In cultured cells and a mouse xenograft model, miR-148a reduced the growth, epithelial-to-mesenchymal transition, invasion, and metastasis of HBx-expressing hepatocarcinoma cells through inhibition of HPIP-mediated mTOR signaling. Thus, miR-148a activation or HPIP inhibition may be a useful strategy for cancer treatment.

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-β–like signaling pathway

The four-and-a-half LIM (FHL) proteins belong to a family of LIM-only proteins that regulate cell proliferation, differentiation, and apoptosis. The exact functions of each FHL protein in cancer development and progression remain unknown. Here we report that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, in a TGF-beta-independent manner. Casein kinase 1delta, but not the TGF-beta receptor, was required for the FHL-mediated TGF-beta-like responses, including increased phosphorylation of Smad2/3, interaction of Smad2/3 and Smad4, nuclear accumulation of Smad proteins, activation of the tumor suppressor gene p21, and repression of the oncogene c-myc. FHL1-3 inhibited anchorage-dependent and -independent growth of a human hepatoma cell line in vitro and tumor formation in nude mice. Further analysis of clinical samples revealed that FHL proteins are often downregulated in hepatocellular carcinomas and that this correlates with decreased TGF-beta-like responses. By establishing a link between FHL proteins and Smad proteins, this study identifies what we believe to be a novel TGF-beta-like signaling pathway and indicates that FHL proteins may be useful molecular targets for cancer therapy.

BRCA1 interacts with FHL2 and enhances FHL2 transactivation function

Germ‐line mutations in BRCA1 are associated with an increased lifetime risk of developing breast and/or ovarian tumors. The BRCA1 gene product is a 220‐kDa protein that contains a tandem of two BRCA1 C‐terminal (BRCT) domains required for transcription. In an attempt to understand how BRCA1 exerts its function through BRCT domains, we search for partners of the BRCT domains of BRCA1. Using the yeast two‐hybrid system, we identified the four and a half LIM‐only protein 2 (FHL2) as a novel BRCA1 interacting protein. We demonstrate that BRCA1 and FHL2 can physically associate in vitro, in yeast, and in human cells. BRCA1 interacted with FHL2 through its second BRCT domain and the interaction of FHL2 with BRCA1 requires the last three LIM domains of FHL2. BRCA1 enhanced FHL2‐mediated transcriptional activity in transient transfections. Tumor‐derived transactivation‐deficient BRCA1 mutants showed a reduced ability to enhance transactivation by FHL2. Lack of BRCA1 binding sites in the FHL2 completely abolished the FHL2 transactivation function. Reverse transcription polymerase chain reaction analysis showed that FHL2 mRNA levels may be downregulated in many breast cancer cell lines. These results suggest that the BRCA1–FHL2 interaction may be involved in transcriptional regulation and play a significant role in cancer cell growth.

Stimulatory cross-talk between NFAT3 and estrogen receptor in breast cancer cells.

Estrogen receptors (ERα and ERβ) are ligand-regulated transcription factors that play critical roles in the development and progression of breast cancer by regulating target genes involved in cellular proliferation. The transcriptional activity of ERα and ERβ is known to be modulated by cofactor proteins. We used a yeast two-hybrid system and identified NFAT3 as a novel ERβ-binding protein. NFAT3 interacted with ERα and ERβ both in vitro and in mammalian cells in a ligand-independent fashion. NFAT3 bound specifically to the ERβ region containing the activation function-1 domain, a ligand-independent transactivation domain. Overexpression of NFAT3 enhanced both ERα and ERβ transcriptional activities in a ligand-independent manner and up-regulated downstream estrogen-responsive genes including pS2 and cathepsin D. Reduction of endogenous NFAT3 with NFAT3 small interfering RNA or overexpression of NFAT3 deletion mutants that lack the ER-binding sites reduced the NFAT3 coactivation of ERα and ERβ. NFAT3 increased binding of ERα to the estrogen-responsive element and was recruited to endogenous estrogen-responsive promoters. NFAT3 was expressed differentially in many breast cancer cell lines and overexpressed in a subset of breast cancer patients. Knockdown of endogenous NFAT3 reduced the growth of human breast cancer ZR75-1 cells in a ligand-independent manner. Taken together, these results suggest that NFAT3 may play important roles in ER signaling and represent a novel target for breast cancer therapy.

PES1 promotes breast cancer by differentially regulating ERα and ERβ

The initiation of breast cancer is associated with increased expression of tumor-promoting estrogen receptor α (ERα) protein and decreased expression of tumor-suppressive ERβ protein. However, the mechanism underlying this process is unknown. Here we show that PES1 (also known as Pescadillo), an estrogen-inducible protein that is overexpressed in breast cancer, can regulate the balance between ERα and ERβ. We found that PES1 modulated many estrogen-responsive genes by enhancing the transcriptional activity of ERα while inhibiting transcriptional activity of ERβ. Consistent with this regulation of ERα and ERβ transcriptional activity, PES1 increased the stability of the ERα protein and decreased that of ERβ through the ubiquitin-proteasome pathway, mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). Moreover, PES1 transformed normal human mammary epithelial cells and was required for estrogen-induced breast tumor growth in nude mice. Further analysis of clinical samples showed that expression of PES1 correlated positively with ERα expression and negatively with ERβ expression and predicted good clinical outcome in breast cancer. Our data demonstrate that PES1 contributes to breast tumor growth through regulating the balance between ERα and ERβ and may be a better target for the development of drugs that selectively regulate ERα and ERβ activities.

A phosphotyrosine switch determines the antitumor activity of ERβ

Estrogen receptors ERα and ERβ share considerable sequence homology yet exert opposite effects on breast cancer cell proliferation. While the proliferative role of ERα in breast tumors is well characterized, it is not clear whether the antitumor activity of ERβ can be mobilized in breast cancer cells. Here, we have shown that phosphorylation of a tyrosine residue (Y36) present in ERβ, but not in ERα, dictates ERβ-specific activation of transcription and is required for ERβ-dependent inhibition of cancer cell growth in culture and in murine xenografts. Additionally, the c-ABL tyrosine kinase and EYA2 phosphatase directly and diametrically controlled the phosphorylation status of Y36 and subsequent ERβ function. A nonphosphorylatable, transcriptionally active ERβ mutant retained antitumor activity but circumvented control by upstream regulators. Phosphorylation of Y36 was required for ERβ-mediated coactivator recruitment to ERβ target promoters. In human breast cancer samples, elevated phosphorylation of Y36 in ERβ correlated with high levels of c-ABL but low EYA2 levels. Furthermore, compared with total ERβ, the presence of phosphorylated Y36-specific ERβ was strongly associated with both disease-free and overall survival in patients with stage II and III disease. Together, these data identify a signaling circuitry that regulates ERβ-specific antitumor activity and has potential as both a prognostic tool and a molecular target for cancer therapy.

The estrogen receptor-interacting protein HPIP increases estrogen-responsive gene expression through activation of MAPK and AKT

Estrogen receptors (ERalpha and ERbeta) are estrogen-regulated transcription factors that play important roles in the development and progression of breast cancer. The biological function of ERs has been shown to be modulated by ER-interacting proteins. However, the ER-interacting proteins that not only activate MAPK and AKT, two important growth regulatory protein kinases, but also increase growth related estrogen-responsive gene expression remain unknown. Here, we report that hematopoietic PBX-interacting protein (HPIP) interacts both with ERalpha and with ERbeta, and increases ERalpha target gene expression through activation of MAPK and AKT and enhanced ERalpha phosphorylation. ERbeta inhibits ERalpha target gene expression, possibly by competition of ERbeta with ERalpha for binding to HPIP, and by a decrease in available ERalpha for HPIP binding through the interaction of ERbeta with ERalpha. Furthermore, HPIP increases breast cancer cell growth. These data suggest that HPIP may be an important regulator in ER signaling and that the relative ratio of ERbeta to ERalpha may be important for HPIP function.

Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells

Hepatitis B virus (HBV) X protein (HBx) is considered to play a role in the development of hepatocellular carcinoma (HCC) during HBV infection. HCC was shown to be more prevalent in men than in women. Estrogen, which exerts its biological function through estrogen receptor (ER), can inhibit HBV replication. ERΔ5, an ERα variant lacking exon 5, was found to be preferentially expressed in patients with HCC compared with patients with normal livers. Here, we report the biological role of ERΔ5 and a novel link between HBx and ERα signaling in hepatoma cells. ERΔ5 interacts with ERα in vitro and in vivo and functions as a dominant negative receptor. Both ERα and ERΔ5 associate with HBx. HBx decreases ERα-dependent transcriptional activity, and HBx and ERΔ5 have additive effect on suppression of ERα transactivation. The HBx deletion mutant that lacks the ERα-binding site abolishes the HBx repression of ERα. HBx, ERα and histone deacetylase 1 (HDAC1) form a ternary complex. Trichostatin A, a specific inhibitor of HDAC enzyme, can restore the transcriptional activity of ERα inhibited by HBx. Our data suggest that HBx and ERΔ5 may play a negative role in ERα signaling and that ERα agonists may be developed for HCC therapy.

Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS.

Smad2, Smad3 and Smad4 proteins are considered to be key mediators of transforming growth factor-β (TGF-β) signaling. However, the identities of the Smad partners mediating TGF-β signaling are not fully understood. Here, we show that RNA-binding protein with multiple splicing (RBPMS), a member of the RNA-binding protein family, physically interacts with Smad2, Smad3 and Smad4 both in vitro and in vivo. The presence of TGF-β increases the binding of RBPMS with these Smad proteins. Consistent with the binding results, overexpression of RBPMS enhances Smad-dependent transcriptional activity in a TGF-β-dependent manner, whereas knockdown of RBPMS decreases this activity. RBPMS interacts with TGF-β receptor type I (TβR-I), increases phosphorylation of C-terminal SSXS regions in Smad2 and Smad3, and promotes the nuclear accumulation of the Smad proteins. Moreover, RBPMS fails to enhance the transcriptional activity of Smad2 and Smad3 that lack the C-terminal phosphorylation sites. Our data provide the first evidence for an RNA-binding protein playing a role in regulation of Smad-mediated transcriptional activity and suggest that RBPMS stimulates Smad-mediated transactivation possibly through enhanced phosphorylation of Smad2 and Smad3 at the C-terminus and promotion of the nuclear accumulation of the Smad proteins.

Downregulation and growth inhibitory role of FHL1 in lung cancer

Four and a half Lin‐11, Isl‐1, Mac‐3 (LIM) protein 1 (FHL1) has been linked to carcinogenesis. However, the role of FHL1 in lung cancer remains unclear and the detailed mechanism underlying its tumor suppressive role is poorly understood. The purpose of this study was to examine FHL1 expression in lung cancer patients and to investigate how it was associated with lung cancer cell growth. Immunoblotting and immunohistochemistry showed that FHL1 protein was downregulated in over 90% of 80 lung cancer patients. FHL1 expression was strongly correlated with tumor histological types (p < 10−4) and the differentiation of the tumor (p = 0.002). FHL1 inhibited anchorage‐dependent and ‐independent growth of human lung cancer cell lines. The inhibitory effects of FHL1 on lung cancer cell growth were associated with both the G1 and the G2/M cell cycle arrest concomitant with a marked inhibition of cyclin A, cyclin B1 and cyclin D as well as the induction of the cyclin dependent kinase inhibitors p21 (WAF1/CIP1) and p27 (Kip1). Direct intratumoral injection of an adenovirus expressing FHL1 dramatically suppressed the growth of A549 lung cancer cells in nude mice. Our data suggest that reduced expression of FHL1 may play an important role in the development and progression of lung cancer and that FHL1 may be a useful target for lung cancer gene therapy.