Mickey C T Hu

Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we show that embryos with a targeted disruption of CARM1 are small in size and die perinatally. The methylation of two known CARM1 substrates, poly(A)-binding protein (PABP1) and the transcriptional cofactor p300, was abolished in knockout embryos and cells. However, CARM1-dependent methylation of histone H3 was not observed. Furthermore, estrogen-responsive gene expression was aberrant in Carm1–/– fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. These findings provide genetic evidence for the essential role of CARM1 in estrogen-mediated transcriptional activation.

High tumoral maspin expression is associated with improved survival of patients with oral squamous cell carcinoma

Maspin, a member of the serpin family of protease inhibitors, is known to have tumor-suppressor functions. However, the association between its expression level and survival has not been demonstrated in human cancer. Using the immunohistochemical technique to examine the expression levels of maspin in 44 cases of oral squamous cell carcinoma (SCC), we found that 66% of the cases expressed low to intermediate levels of maspin and 34% of the cases expressed high levels of maspin. We further examined maspin protein expression in a series of six SCC cell lines from the head and neck, and found that all but one expressed low or no maspin protein. We also compared the clinicopathological features of the oral SCC cases with the maspin expression level, and found that high maspin expression was associated with the absence of lymph node metastasis. More importantly, we showed that higher maspin expression was significantly associated with better rates of overall survival, suggesting that high maspin expression may be a favorable prognostic marker for oral SCC.

Functional interaction between FOXO3a and ATM regulates DNA damage response

The maintenance of genomic stability in cells is relentlessly challenged by environmental stresses that induce DNA breaks, which activate the DNA-damage pathway mediated by ataxia-telangiectasia mutated (ATM) and its downstream mediators to control damage-induced cell-cycle checkpoints and DNA repair. Here, we show that FOXO3a interacts with ATM to promote phosphorylation of ATM at Ser 1981 and prompting its downstream mediators to form nuclear foci in response to DNA damage. Silencing FOXO3a in cells abrogates the formation of ATM-pS1981 and phospho-histone H2AX foci after DNA damage. Increasing FOXO3a in cells promotes ATM-regulated signalling, the intra-S-phase or G2–M cell-cycle checkpoints, and the repair of damaged DNA, whereas cells lacking FOXO3a did not trigger the DNA-repair mechanism after DNA damage. The carboxy-terminal domain of FOXO3a binds to the FAT domain of ATM, thereby contributing to the activation of ATM. These results suggest that ATM may be regulated directly by FOXO3a in the DNA-damage response.

Cadherin-11 Promotes the Metastasis of Prostate Cancer Cells to Bone

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis–derived PC3 cells with cadherin-11–specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone. (Mol Cancer Res 2008;6(8):1259–67)

JNK1, JNK2 and JNK3 are p53 N-terminal serine 34 kinases.

The function of the tumor suppressor protein p53 is modulated by post-translational events, primarily by phosphorylation. p53 is phosphorylated at multiple sites by a variety of protein kinases depending on the cellular environment. It has been suggested that serine 34 of mouse p53 is specifically phosphorylated by a stress- activated protein kinase in response to ultraviolet radiation. Since serine 34 is a major site of phosphorylation of mouse p53 in vivo and its specific protein kinase is still not definitively identified yet, we have examined the c-Jun N-terminal kinase 1 (JNK1) activity on p53 by expressing JNK1 in 293T cells. We show here that activated JNK1 phosphorylates mouse p53 specifically at serine 34 in vitro, while a dominanant-negative JNK1 mutant does not phosphorylate p53. More importantly, JNK1 associates with p53 in vivo, with or without activation, confirming that JNK1 is indeed a p53 kinase. Interestingly, activated JNK2 and JNK3 also phosphorylate serine 34 of mouse p53. Furthermore, JNK2 and JNK3 also associate with p53 in vivo, indicating that not only JNK1, but also JNK2 and JNK3 are p53 N-terminal serine 34 kinases. Phosphorylation of p53 by JNKs may play an important role in nuclear signal transduction in response to environmental stress or tumorigenic agents.

E1A Sensitizes Cells to Tumor Necrosis Factor-induced Apoptosis through Inhibition of IκB Kinases and Nuclear Factor κB Activities

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor κB (NF-κB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-κB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-κB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IκB kinase (IKK) activity and IκB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IκB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-κB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.

Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis

IntroductionEstrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. Identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The forkhead box class O (FOXO) transcription factor FOXO3a has been suggested to function as a tumor suppressor in breast cancer. Using protein-protein interaction screening, we found that FOXO3a interacted with ER-α and ER-β proteins in the human breast carcinoma cell line MCF-7, suggesting that there exists a crosstalk between the FOXO3a and ER signaling pathways in estrogen-dependent breast cancer cells.MethodsThe interaction between FOXO3a and ER was investigated by using co-immunoprecipitation and immunoblotting assays. Inhibition of ER-α and ER-β transactivation activity by FOXO was determined by luciferase reporter assays. Cell proliferation in culture was evaluated by counting cell numbers. Tumorigenesis was assessed in athymic mice that were injected with MCF-7 cell lines over-expressing FOXO3a. Protein expression levels of cyclin-dependent kinase inhibitors, cyclins, ERs, FOXM1, and the proteins encoded by ER-regulated genes in MCF-7 cell lines and breast tumors were examined by immunoblotting analysis and immunohistochemical staining.ResultsWe found that FOXO3a interacted with ER-α and ER-β proteins and inhibited 17β-estradiol (E2)-dependent, ER-regulated transcriptional activities. Consistent with these observations, expression of FOXO3a in the ER-positive MCF-7 cells decreased the expression of several ER-regulated genes, some of which play important roles in cell proliferation. Moreover, we found that FOXO3a upregulated the expression of the cyclin-dependent kinase inhibitors p21Cip1, p27Kip1, and p57Kip2. These findings suggest that FOXO3a induces cell growth arrest to effect tumor suppression. FOXO3a repressed the growth and survival of MCF-7 cells in cell culture. In an orthotopic breast cancer xenograft model in athymic mice, over-expression of FOXO3a in MCF-7 cells suppressed their E2-induced tumorigenesis, whereas knockdown of FOXO3a in MCF-7 resulted in the E2-independent growth.ConclusionFunctional interaction between FOXO3a and ER plays a critical role in suppressing estrogen-dependent breast cancer cell growth and tumorigenesis in vivo. This suggests that agents that activate FOXO3a may be novel therapeutic agents that can inhibit and prevent tumor proliferation and development in breast cancer.

Hematopoietic progenitor kinase-1 (HPK1) stress response signaling pathway activates IκB kinases (IKK-α/β) and IKK-β is a developmentally regulated protein kinase

Nuclear factor kappa-B (NF-κB) is a pleiotropic transcription factor that plays a central role in the immune and inflammatory responses, and is also involved in controlling viral transcription and apoptosis. A critical control in the activation of NF-κB is the phosphorylation of its inhibitory factor IκBs by IκB kinases (IKK-α and -β). Here, we present experiments addressing the regulation and global expression of murine IKK-β, and localize the IKK-β gene to mouse chromosome 8A3-A4. IKK-β was expressed primarily in the liver, kidney and spleen, and at lower levels in the other adult tissues. While IKK-β was expressed ubiquitously throughout the mouse embryo at 9.5 days, its expression began to be localized to the brain, neural ganglia, neural tube, and liver in the 12.5-days embryo. At 15.5 days, the expression of IKK-β was further restricted to specific tissues of the embryo, suggesting that IKK-β is a developmentally regulated protein kinase. Interestingly, IKK-β phosphorylated IκB constitutively, whereas IKK-α was not active in the absence of cell stimulation. Moreover, both IKK-α and -β were activated by hematopoietic progenitor kinase-1 (HPK1) and MAPK/ERK kinase kinase-1 (MEKK1) specifically, suggesting that IκB/NF-κB is regulated through the HPK1-MEKK1 stress response signaling pathway.

Assessment of epidermal growth factor receptor with 99mTc-ethylenedicysteine-C225 monoclonal antibody.

Epidermal growth factor receptor (EGFR) plays an important role in cell division and cancer progression, as well as angiogenesis and metastasis. Since many tumor cells exhibit the EGFR on their surface, functional imaging of EGFR provides not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-EGFR therapy could be assessed effectively. C225 is a chimeric monoclonal antibody that targets the human extracellular EGFR and inhibits the growth of EGFR-expressing tumor cells. Also, it has been demonstrated that C225, in combination with chemotherapeutic drugs or radiotherapy, is effective in eradicating well-established tumors in nude mice. We have developed 99mTc-labeled C225 using ethylenedicysteine (EC) as a chelator. This study aimed at measuring uptake of 99mTc–EC–C225 in EGFR+ tumor-bearing animal models and preliminary feasibility of imaging patients with head and neck carcinomas. In vitro Western blot analysis and cytotoxicity assays were used to examine the integrity of EC–C225. Tissue distribution studies of 99mTc–EC–C225 were evaluated in tumor-bearing rodents at 0.5–4 h. In vivo biodistribution of 99mTc–EC–C225 in tumor-bearing rodents showed increased tumor-to-tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using 99mTc–EC–C225 to assess EGFR expression. SPECT images confirmed that the tumors could be visualized with 99mTc–EC–C225 from 0.5 to 4 h in tumor bearing rodents. We conclude that 99mTc–EC–C225 may be useful to assess tumor EGFR expression. This may be useful in the future for selecting patients for treatment with C225.

Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone. (Mol Cancer Res 2007;5(7):675–84)