Chaeyong Jung

HOXB13 Induces Growth Suppression of Prostate Cancer Cells as a Repressor of Hormone-Activated Androgen Receptor Signaling

Androgen receptor (AR) signals play a decisive role in regulating the growth and differentiation of both normal and cancerous prostate cells by triggering the regulation of target genes, in a process in which AR cofactors have critical functions. Because of the highly prostate-specific expression pattern of HOXB13, we studied the role of this homeodomain protein in prostate cells. Expression of HOXB13 was limited to AR-expressing prostate cells. Reporter transcription assay demonstrated that HOXB13 significantly suppressed hormone-mediated AR activity in a dose-responsive manner, and suppression was specific to AR with which HOXB13 physically interacts. Overexpression of HOXB13 further down-regulated the androgen-stimulated expression of prostate-specific antigen, and suppression of endogenous HOXB13 stimulated transactivation of AR. Functionally, HOXB13 suppressed growth of LNCaP prostate cancer cells, which could be counteracted by additional hormone-activated AR. On the other hand, the growth-suppressive function of HOXB13 in AR-negative CV-1 cells was not affected by AR. These results suggest that HOXB13 functions as an AR repressor to modulate the complex AR signaling and subsequent growth regulation of prostate cancer cells. In addition to the loss of HOXB13 expression, maintaining AR may be an important step for prostate cancer cells to tolerate the suppressor function of HOXB13. Altogether, our data present a novel mechanism for the HOXB13-mediated repression of AR signaling, which can be interpreted to a growth-suppressive event.

HOXB13 homeodomain protein suppresses the growth of prostate cancer cells by the negative regulation of T-cell factor 4

In prostate gland, HOXB13 is highly expressed from the embryonic stages to adulthood. However, the function of HOXB13 in normal cell growth and tumorigenesis is not yet known. We investigated the role of HOXB13 and mechanism by which it functions in HOXB13-negative cells. Expression of HOXB13 was forced in HOXB13-negative PC3 prostate cancer cells using a liposome-mediated gene transfer approach. Compared with the control clones, HOXB13-expressing PC3 cells exhibited significant inhibition of in vitro and in vivo cell growth with G1 cell cycle arrest mediated by the suppression of cyclin D1 expression. Because cyclin D1 is mainly regulated by beta-catenin/T-cell factor (TCF), TCF-4 response element was used in a reporter gene transcription assay, demonstrating that HOXB13 significantly inhibits TCF-4-mediated transcriptional activity in both prostate and nonprostate cells. This inhibition occurred in a dose-responsive manner and was specific to TCF-4 response element. Western blot analysis demonstrated that HOXB13 down-regulates the expression of TCF-4 and its responsive genes, c-myc and cyclin D1. HOXB13 also suppressed the activity of natural c-myc promoter. This study suggests that HOXB13, a transcription factor, functions as a cell growth suppressor by negatively regulating the expression of TCF-4, which eventually provides negative signals for cell proliferation. This observation will provide valuable insight into the molecular basis of prostate tumorigenesis.

Gene Therapy for Prostate Cancer by Controlling Adenovirus E1a and E4 Gene Expression with PSES Enhancer

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.

Differential CARM1 expression in prostate and colorectal cancers

BackgroundCoactivator-associated arginine methyltransferase 1 (CARM1) functions as a transcriptional coactivator of androgen receptor (AR)-mediated signaling. Correspondingly, overexpression of CARM1 has been associated with the development of prostate cancer (PCa) and its progression to androgen-independent PCa. In our preliminary study, however, the promoting effects of CARM1, with regard to androgen-stimulated AR target gene expression were minimal. These results suggested that the AR target gene expression associated with CARM1 may result primarily from non-hormone dependent activity. The goal of this study was to confirm the pattern of expression of CARM1 in human tumors and determine the mechanism of action in CARM1 overexpressed tumors.MethodsTissue microarray was used to determine the pattern of expression of CARM1 in human cancers by immunohistochemistry. CARM1 expression was also evaluated in prostate and colorectal surgical specimens and the clinical records of all cases were reviewed. In addition, a reporter transcription assay using the prostate-specific antigen (PSA) promoter was used to identify the signaling pathways involved in non-hormone-mediated signal activation associated with CARM1.ResultsThe tissue microarray showed that CARM1 was particularly overexpressed in the colorectal cancers while CARM1 expression was not prevalent in the prostate and breast cancers. Further studies using surgical specimens demonstrated that CARM1 was highly overexpressed in 75% of colorectal cancers (49 out of 65) but not in the androgen-independent PCa. In addition, CARM1s coactivating effect on the entire PSA promoter was very limited in both androgen-dependent and androgen-independent PCa cells. These results suggest that there are other factors associated with CARM1 expression in PSA regulation. Indeed, CARM1 significantly regulated both p53 and NF-κB target gene transcription.ConclusionsThe results of this study suggest that, in addition to its role in activation of steroid receptors, CARM1 functions as a transcriptional modulator by altering the activity of many transcriptional factors, especially with regard to androgen independent PCa and colorectal cancers.

HOXB13 promotes androgen independent growth of LNCaP prostate cancer cells by the activation of E2F signaling.

BackgroundAndrogen signaling plays a critical role in the development of prostate cancer and its progression. However, androgen-independent prostate cancer cells emerge after hormone ablation therapy, resulting in significant clinical problems. We have previously demonstrated that the HOXB13 homeodomain protein functions as a prostate cancer cell growth suppressor by inhibiting androgen-mediated signals. However, the role of the HOXB13 in androgen-independent growth of prostate cancer cells remains unexplained.ResultsIn this report, we first demonstrated that HOXB13 was highly overexpressed in hormone-refractory tumors compared to tumors without prostate-specific antigen after initial treatment. Functionally, in an androgen-free environment minimal induction of HOXB13 in LNCaP prostate cancer cells, to the level of the normal prostate, markedly promoted cell proliferation while suppression inhibited cell proliferation. The HOXB13-mediated cell growth promotion in the absence of androgen, appears to be mainly accomplished through the activation of RB-E2F signaling by inhibiting the expression of the p21waf tumor suppressor. Indeed, forced expression of HOXB13 dramatically decreased expression of p21waf; this inhibition largely affected HOXB13-mediated promotion of E2F signaling.ConclusionsTaken together, the results of this study demonstrated the presence of a novel pathway that helps understand androgen-independent survival of prostate cancer cells. These findings suggest that upregulation of HOXB13 is associated with an additive growth advantage of prostate cancer cells in the absence of or low androgen concentrations, by the regulation of p21-mediated E2F signaling.

δ-Catenin promotes E-cadherin processing and activates β-catenin-mediated signaling: implications on human prostate cancer progression.

δ-Catenin binds the juxtamembrane domain of E-cadherin and is known to be overexpressed in some human tumors. However, the functions of δ-catenin in epithelial cells and carcinomas remain elusive. We found that prostate cancer cells overexpressing δ-catenin show an increase in multi-layer growth in culture. In these cells, δ-catenin colocalizes with E-cadherin at the plasma membrane, and the E-cadherin processing is noticeably elevated. E-Cadherin processing induced by δ-catenin is serum-dependent and requires MMP- and PS-1/γ-secretase-mediated activities. A deletion mutant of δ-catenin that deprives the ability of δ-catenin to bind E-cadherin or to recruit PS-1 to E-cadherin totally abolishes the δ-catenin-induced E-cadherin processing and the multi-layer growth of the cells. In addition, prostate cancer cells overexpressing δ-catenin display an elevated total β-catenin level and increase its nuclear distribution, resulting in the activation of β-catenin/LEF-1-mediated transcription and their downstream target genes as well as androgen receptor-mediated transcription. Indeed, human prostate tumor xenograft in nude mice, which is derived from cells overexpressing δ-catenin, shows increased β-catenin nuclear localization and more rapid growth rates. Moreover, the metastatic xenograft tumor weights positively correlate with the level of 29kD E-cadherin fragment, and primary human prostate tumor tissues also show elevated levels of δ-catenin expression and the E-cadherin processing. Taken together, these results suggest that δ-catenin plays an important role in prostate cancer progression through inducing E-cadherin processing and thereby activating β-catenin-mediated oncogenic signals.

HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression.

During the prostate cancer (PCa) development and its progression into hormone independency, androgen receptor (AR) signals play a central role by triggering the regulation of target genes, including prostate-specific antigen. However, the regulation of these AR-mediated target genes is not fully understood. We have previously demonstrated a unique role of HOXB13 homeodomain protein as an AR repressor. Expression of HOXB13 was highly restricted to the prostate and its suppression dramatically increased hormone-activated AR transactivation, suggesting that prostate-specific HOXB13 was a highly potent transcriptional regulator. In this report, we demonstrated the action mechanism of HOXB13 as an AR repressor. HOXB13 suppressed androgen-stimulated AR activity by interacting with AR. HOXB13 did neither bind to AR responsive elements nor disturb nuclear translocation of AR in response to androgen. In PCa specimen, we also observed mutual expression pattern of HOXB13 and AR. These results suggest that HOXB13 not only serve as a DNA-bound transcription factor but play an important role as an AR-interacting repressor to modulate hormone-activated androgen receptor signals. Further extensive studies will uncover a novel mechanism for regulating AR-signaling pathway to lead to expose new role of HOXB13 as a non-DNA-binding transcriptional repressor.

Anti‐tumor efficacy of a transcriptional replication‐competent adenovirus, Ad‐OC‐E1a, for osteosarcoma pulmonary metastasis

Osteosarcoma (OSA) is the most frequent type of primary malignant bone tumor and is apt to occur in children and young adults. Pulmonary metastasis (OSPM) is the major reason for its fatal outcome. Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to bone marrow and osteotropic tumors. OC is also known to express in cell lines with bone metastasis feathers. Gene therapy strategies with the OC promoter directing the replication of adenovirus in a tumor‐specific manner are a potential modality for OSPM therapy.

Bilateral asymmetric supernumerary heads of biceps brachii.

Anatomical variations of the biceps brachii have been described by various authors, but the occurrence of bilateral asymmetric supernumerary heads is rare and has not been reported. We found three accessory heads of the biceps brachii muscle on right arm and an anomalous third head of biceps brachii on left arm. The third, fourth, and fifth heads of right arm originated from the body of humerus at the insertion site of coracobrachialis and inserted into the distal part of biceps brachii short head in order. The third head of left arm originated from humerus at the insertion site of coracobrachialis and combined with the distal part of biceps brachii and continued to the proximal part of common biceps tendon. Understanding the existence of bilateral asymmetric supernumerary heads of biceps brachii may influence preoperative diagnosis and surgery on the upper limbs.

Evaluation of HOXB13 as a molecular marker of recurrent prostate cancer

Many patients with prostate cancer have disease recurrence following surgical removal of tumors and fail to respond to androgen ablation therapy. Despite the existence of a number of clinical/pathological factors, it is not possible to predict which patients will fall into this category. The results of our previous studies demonstrated that the HOXB13 homeodomain protein plays a key role in the development of prostate cancer and the progression of this malignancy. In addition, HOXB13 has been reported to predict estrogen-resistant breast cancer tumors. The purpose of this study was to investigate whether HOXB13 could be used as a molecular marker to predict prostate cancer recurrence. To examine the role of HOXB13 as a molecular marker with clinical/pathological data, the expression of HOXB13 was compared using immunohistochemistry in 57 organ-confined prostate cancer tumors obtained by radical prostatectomy. There was no significant correlation between the expression of HOXB13 and most clinical/pathological parameters, including tumor margin, invasion, pathological stage and risk level. The HOXB13 expression levels correlated with the Gleason score and there was a positive correlation with the pre-operative prostate specific antigen (PSA) levels. Accordingly, the tumor specimens from 4 patients who ultimately had biochemical failure (PSA >0.2 ng/ml), all showed a high expression of HOXB13, while their risk levels were either intermediate or high. This is the first study to report that HOXB13, together with other clinical/pathological factors, can be used as a molecular marker to predict the progression of prostate cancer.