Targeting SOX4/PCK2 signaling suppresses neuroendocrine trans-differentiation of castration-resistant prostate cancer.

BACKGROUND: Neuroendocrine prostate cancer (NEPC), a lethal subset of prostate cancer (PCa), is characterized by loss of AR signaling and resistance to AR-targeted therapy. While it is well reported that second-generation AR blockers induce neuroendocrine (NE) trans-differentiation of castration-resistant prostate cancer (CRPC) to promote the occurrence of NEPC, and pluripotent transcription factors might be potential regulators, the underlying molecular mechanisms remain unclear. METHODS: We analyzed the data from public databsets to screen candidate genes and then focused on SOX4, a regulator of NE trans-differentiation. The expression changes of SOX4 and its relationship with tumor progression were validated in clinical tumor tissues. We evaluated malignant characteristics related to NEPC in prostate cancer cell lines with stable overexpression or knockdown of SOX4 in vitro. Tumor xenografts were analyzed after inoculating the relevant cell lines into nude mice. RNA-seq, ATAC-seq, non-targeted metabolomics analysis, as well as molecular and biochemical assays were carried out to determine the mechanism. RESULTS: We screened public datasets and identified that expression of SOX4 was significantly elevated in NEPC. Overexpressing SOX4 in C4-2B cells increased cell proliferation and migration, upregulated the expression of NE marker genes, and inhibited AR expression. Consistently, inhibition of SOX4 expression in DU-145 and PC-3 cells reduced the above malignant phenotypes and repressed the expression of NE marker genes. For the in vivo assay, we found that knockdown of SOX4 inhibited tumor growth of subcutaneous xenografts in castrated nude mice which were concomitantly treated with enzalutamide (ENZ). Mechanically, we identified that one of the key enzymes in gluconeogenesis, PCK2, was a novel target of SOX4. The activation of carbohydrate metabolism reprogramming by SOX4 could promote NE trans-differentiation via the SOX4/PCK2 pathway. CONCLUSIONS: Our findings reveal that SOX4 promotes NE trans-differentiation both in vitro and in vivo via directly enhancing PCK2 activity to activate carbohydrate metabolism reprogramming. The SOX4/PCK2 pathway and its downstream changes might be novel targets for blocking NE trans-differentiation.

PAX6 promotes neuroendocrine phenotypes of prostate cancer via enhancing MET/STAT5A-mediated chromatin accessibility.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a lethal subset of prostate cancer which is characterized by neuroendocrine differentiation and loss of androgen receptor (AR) signaling. Growing evidence reveals that cell lineage plasticity is crucial in the failure of NEPC therapies. Although studies suggest the involvement of the neural transcription factor PAX6 in drug resistance, its specific role in NEPC remains unclear. METHODS: The expression of PAX6 in NEPC was identified via bioinformatics and immunohistochemistry. CCK8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay were used to illustrate the key role of PAX6 in the progression of in vitro. ChIP and Dual-luciferase reporter assays were conducted to confirm the binding sequences of AR in the promoter region of PAX6, as well as the binding sequences of PAX6 in the promoter regions of STAT5A and MET. For in vivo validation, the xenograft model representing NEPC subtype underwent pathological analysis to verify the significant role of PAX6 in disease progression. Complementary diagnoses were established through public clinical datasets and transcriptome sequencing of specific cell lines. ATAC-seq was used to detect the chromatin accessibility of specific cell lines. RESULTS: PAX6 expression was significantly elevated in NEPC and negatively regulated by AR signaling. Activation of PAX6 in non-NEPC cells led to NE trans-differentiation, while knock-down of PAX6 in NEPC cells inhibited the development and progression of NEPC. Importantly, loss of AR resulted in an enhanced expression of PAX6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the MET/STAT5A signaling pathway. Through ATAC-seq, we found that a high expression level of PAX6 elicited enhanced chromatin accessibility, mainly through attenuation of H4K20me3, which typically causes chromatin silence in cancer cells. CONCLUSION: This study reveals a novel neural transcription factor PAX6 could drive NEPC progression and suggest that it might serve as a potential therapeutic target for the management of NEPC.

Taurine Inhibits Ferroptosis Mediated by the Crosstalk between Tumor Cells and Tumor-Associated Macrophages in Prostate Cancer.

Tumor-associated macrophages (TAMs) play an essential role in tumor therapeutic resistance. Although the lethal effect of ferroptosis on tumor cells is well reported, how TAMs inhibit the effect of ferroptosis in tumors has not been clearly defined. In this study, it is demonstrated that TAM-secreted taurine suppresses ferroptosis in prostate cancer (PCa) by activating the Liver X receptor alpha/Stearoyl-Coenzyme A desaturase 1 (LXRα/SCD1) pathway. Blocking taurine intake via inhibition of taurine transporter TauT restores the sensitivity to ferroptosis in tumors. Furthermore, LXRα activates the transcription of both miR-181a-5p and its binding protein FUS to increase the recruitment of miR-181a-5p in tumor-derived extracellular vesicles (EVs). It is observed that macrophages appear to be recipient cells of the miR-181a-5p-enriched EVs. Intake of miR-181a-5p in macrophages promotes their M2 polarization and enhances the taurine export by inhibiting expression of its target gene lats1, which in turn inactivates the hippo pathway and results in a Yes-associated protein (YAP) nuclear translocation for transcriptional activation of both M2 polarization-related genes such as ARG1 and CD163 and the taurine transport gene TauT. Taken together, the findings indicate a reciprocal interaction between PCa cells and TAMs as a positive feedback-loop to repress ferroptosis in PCa, mediated by TAM-secreted taurine and tumor EV-delivered miR-181a-5p.

Discovery of extracellular vesicle-delivered miR-185-5p in the plasma of patients as an indicator for advanced adenoma and colorectal cancer.

BACKGROUND: We aimed to evaluate whether extracellular vesicles (EV)-derived microRNAs (miRNAs) can be used as biomarkers for advanced adenoma (AA) and colorectal cancer (CRC). METHODS: We detected the changes in the plasma EV-delivered miRNA profiles in healthy donor (HD), AA patient, and I-II stage CRC patient groups using miRNA deep sequencing assay. We performed the TaqMan miRNA assay using 173 plasma samples (two independent cohorts) from HDs, AA patients, and CRC patients to identify the candidate miRNA(s). The accuracy of candidate miRNA(s) in diagnosing AA and CRC was determined using the area under the receiver-operating characteristic curve (AUC) values. Logistic regression analysis was performed to evaluate the association of candidate miRNA(s) as an independent factor for the diagnosis of AA and CRC. The role of candidate miRNA(s) in the malignant progression of CRC was explored using functional assays. RESULTS: We screened and identified four prospective EV-delivered miRNAs, including miR-185-5p, which were significantly upregulated or downregulated in AA vs. HD and CRC vs. AA groups. In two independent cohorts, miR-185-5p was the best potential biomarker with the AUCs of 0.737 (Cohort I) and 0.720 (Cohort II) for AA vs. HD diagnosis, 0.887 (Cohort I) and 0.803 (Cohort II) for CRC vs. HD diagnosis, and 0.700 (Cohort I) and 0.631 (Cohort II) for CRC vs. AA diagnosis. Finally, we demonstrated that the upregulated expression of miR-185-5p promoted the malignant progression of CRC. CONCLUSION: EV-delivered miR-185-5p in the plasma of patients is a promising diagnostic biomarker for colorectal AA and CRC. Trial registration The study protocol was approved by the Ethics Committee of Changzheng Hospital, Naval Medical University, China (Ethics No. 2022SL005, Registration No. of China Clinical Trial Registration Center: ChiCTR220061592).

Tumor-derived miR-378a-3p-containing extracellular vesicles promote osteolysis by activating the Dyrk1a/Nfatc1/Angptl2 axis for bone metastasis.

Most prostate cancer (PCa)-related deaths are caused by progression to bone metastasis. Recently, the importance of extracellular vesicles (EVs) in pre-metastatic niche formation has been reported. However, whether and how tumor-derived EVs interact with bone marrow macrophages (BMMs) to release EV-delivered microRNAs to promote osteolysis and induce pre-metastatic niche formation for PCa bone metastasis remain unclear. Our in vitro and in vivo functional and mechanistic assays revealed that EV-mediated release of miR-378a-3p from tumor cells was upregulated in bone-metastatic PCa, maintaining low intracellular miR-378a-3p concentration to promote proliferation and MAOA-mediated epithelial-to-mesenchymal transition. Moreover, miR-378a-3p enrichment in tumor-derived EVs was induced by hnRNPA2B1 (a transfer chaperone) overexpression. After tumor-derived EVs were taken in by BMMs, enriched miR-378a-3p promoted osteolytic progression by inhibiting Dyrk1a to improve Nfatc1 (an osteolysis-related transcription factor) nuclear translocation, to activate the expression of downstream target gene Angptl2. As a feedback, increased Angptl2 secretion into the tumor environment promoted PCa progression. In conclusion, tumor-derived miR-378a-3p-containing EVs play a significant role in PCa bone metastasis by activating the Dyrk1a/Nfatc1/Angptl2 axis in BMMs to induce osteolytic progression, making miR-378a-3p a potential predictor of metastatic PCa. Reducing the release of miR-378a-3p-containing EVs or inhibiting the recruitment of miR-378a-3p into EVs can be a therapeutic strategy against PCa metastasis.

Regulation of Formation, Stemness and Therapeutic Resistance of Cancer Stem Cells.

Over the past 20 years cancer stem cells (CSCs) have been proposed as key players in the tumorigenesis and progression, which are closely related to the initiation, metastasis and therapeutic resistance of cancer. Evidences have been provided that both genetic and epigenetic factors contribute to the regulation of the formation and stemness maintenance as well as the therapeutic resistance of CSCs via affecting various signal pathways. In addition, the interaction between CSCs and tumor microenvironment has also been revealed to be involved in the above-described processes. With the aim of targeting CSCs to improve treatment outcome, we herein discuss the mechanisms that orchestrate the characteristic of CSCs by the three elements and potential therapeutic strategies. We also summarize how several key regulatory factors function in the regulation of not only the formation and stemness maintenance, but also the therapeutic resistance of CSCs. Thus, future studies focusing on these key factors would be helpful for the development of novel drugs targeting CSCs.

Discovery of extracellular vesicles derived miR-181a-5p in patient's serum as an indicator for bone-metastatic prostate cancer.

Purpose: To identify extracellular vesicle (EV)-delivered microRNAs in the patient's serum as indicators for bone-metastatic prostate cancer. Methods: First, the profiling change of serum EV-delivered miRNAs in patients with either benign prostatic hyperplasia (BPH), non-bone metastatic prostate cancer or bone-metastatic prostate cancer was detected by microRNA deep sequencing assay and microRNA-chip array assay, respectively. Second, the candidates were further confirmed using TaqMan microRNA assay in two independent validation cohorts of total 176 patients with either BPH, non-bone metastatic prostate cancer or bone metastatic prostate cancer to seek the most valuable microRNA(s). Results: Through microRNA deep sequencing and microRNA-chip array, we found 4 prospective EV-delivered miRNAs including miR-181a-5p with significantly upregulated expression in bone metastatic groups than in non-bone metastatic prostate cancer groups (p < 0.05). In the validation cohorts, logistic regression analysis was performed to evaluate the diagnostic association of candidates with bone metastasis, which indicated that miR-181a-5p was significantly associated with bone metastatic prostate cancer. Furthermore, accuracy estimate of each candidate for the diagnosis of bone metastatic prostate cancer was quantified using the area under the receiver-operating characteristic curve (AUC), which identified miR-181a-5p as the best biomarker with the AUCs of 85.6% for diagnosis of prostate cancer and 73.8% for diagnosis of bone metastatic prostate cancer. Conclusion: EV-delivered miR-181a-5p from patient's serum is a promising diagnostic biomarker for bone metastatic prostate cancer.

Elevated expression of Gab1 promotes breast cancer metastasis by dissociating the PAR complex.

BACKGROUND: Breast cancer (BCa) remains as the second leading cause of cancer-related death in women worldwide. The majority of the deaths are due to its progression to metastatic BCa. Although Grb2-associated binding protein 1 (Gab1) has been implicated in tumor proliferation and metastasis in multiple tumors including colorectal cancer, hepatocellular carcinoma and ovarian cancer, whether and how it regulates BCa metastasis are still poorly understood. METHODS: Western blot assay and immunohistochemical (IHC) staining were performed to assess expression of Gab1 in primary and metastatic BCa clinical samples. Biological function assay studies in vitro and in vivo were employed to investigate the functions of Gab1 during BCa metastasis. Co-immunoprecipitation (co-IP) assessment, western blot assay and immunofluorescence (IF) staining were carried out to investigate the underlying mechanism for the function of Gab1 on BCa metastasis. RESULTS: In this study, we found that expression level of Gab1 was increased significantly in BCa tissue samples compared to that in benign mammary hyperplastic tissues. Furthermore, elevated expression of Gab1 was positively associated with metastasis in HER2 and TNBC subtypes of BCa. In BCa cell line MDA-MB-231 and SK-BR3 cells, stable overexpression of Gab1 promoted, while knockdown of Gab1 inhibited cell migration in vitro and metastasis in vivo. Mechanistically, overexpression of Gab1 enhanced its interaction with Par3, a key component of the polarity-associated partitioning defective (PAR) complex, leading to a dissociation of the PAR complex. Consequently, dissociated PAR complex induced epithelial-to-mesenchymal transition (EMT) for breast tumor metastasis. By restoration assessment, we found that only re-expression of a fully functional Gab1, but not a mutant Gab1 that harbors either Par3 binding-deficiency or Par1b binding-deficiency, could reverse the repressive phenotype of cell migration in vitro and metastasis in vivo due to Gab1 knockdown. CONCLUSIONS: Our findings indicate that elevated expression of Gab1 promotes BCa metastasis by dissociating the PAR complex that leads to EMT, implicating a role of Gab1 as a potential biomarker of metastatic BCa. Moreover, inhibition of Gab1 expression might be a promising therapeutic strategy for BCa metastasis.

Loss of Par3 promotes prostatic tumorigenesis by enhancing cell growth and changing cell division modes.

Although cell polarity plays an important role in epithelial tumorigenesis, the consequence of polarity protein loss in prostatic tumorigenesis and the underlying mechanisms remain unclear. Using conditional knockout mouse models, we found in the current study that loss of polarity protein Par3 increases prostatic epithelial cell growth, elevates symmetrical cell divisions in basal cells, and randomizes spindle orientation in luminal cells, causing the development of high-grade prostatic intraepithelial neoplasia (PIN). Mechanistically, loss of Par3 dissociates the Par3/merlin/Lats1 complex, consequently inhibiting phosphorylation of Lats1 to attenuate the Hippo pathway. Furthermore, attenuated Hippo pathway enhances nuclear translocation of Yes-associated protein (YAP), which promotes cell proliferation and symmetrical cell divisions through transcriptional activation of Ki-67 and Sox2. In addition, Lats1 dephosphorylation impairs its interaction with G protein signaling modulator 2 (GPSM2, which is also known as LGN) that causes randomization of spindle orientation in luminal cells. Interestingly, co-deletion of Par3 and Lats1 for complete blockade of the Hippo pathway in mice results in prostate tumor initiation, whereas co-deletion of Par3 and YAP for disrupting YAP nuclear translocation reverses the phenotypes to a relatively normal state. Therefore, our findings highlight combination of Par3 loss and blockade of the Hippo pathway as a novel mechanism for prostatic tumorigenesis.

Pharmacological inhibition of the Notch pathway enhances the efficacy of androgen deprivation therapy for prostate cancer.

Although androgen deprivation therapy (ADT) is a standard treatment for metastatic prostate cancer, this disease inevitably recurs and progresses to ADT-resistant stage after this therapy. Accordingly, understanding the mechanism of resistance to ADT and finding new approach to enhance the efficacy of ADT may provide a major benefit to PCa patients. In our study, we found upregulated expression of Notch receptors is positive associated with ADT-resistance progression. Using fluorescent Notch signaling reporter system, we observed that endogenous Notch signaling could be activated after treatment of androgen deprivation in LNCaP cells via activation of Notch3. In addition, exogenous activation of the Notch signaling though Dox-induced overexpression of any Notch intracellular domains (NICD1-4) could enhance the resistance of PCa cells to ADT under ex vivo 3D culture conditions and upregulate expression of ADT resistance-associated phospho-p38 and Bcl-2 in LNCaP cells. As a result, pharmacological inhibition of the Notch pathway using γ-secretase inhibitor (GSI), DAPT, downregulated both phospho-p38 and Bcl-2 expression and significantly enhanced the efficacy of ADT in androgen sensitive PCa cells with impaired proliferation and 3D colony formation, increased apoptosis and remarkable inhibition of tumor growth in murine subcutaneous xenograft model. These results indicate that activated Notch signaling contributes to ADT resistance, and suggest that inhibition of the Notch pathway may be a promising adjuvant therapy of ADT for PCa.

Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing.

The microRNA-transcription factor auto-regulatory feedback loop is a pivotal mechanism for homeostatic regulation of gene expression, and dysregulation of the feedback loop is tightly associated with tumorigenesis and progression. However, the mechanism underlying such dysregulation is still not well-understood. Here we reported that Krüppel-like factor 4 (KLF4), a stemness-associated transcription factor, promotes the transcription of miR-7 to repress its own translation so that a KLF4-miR-7 auto-regulatory feedback loop is established for mutual regulation of their expression. Interestingly, this feedback loop is unbalanced in prostate cancer (PCa) cell lines and patient samples due to an impaired miR-7-processing, leading to decreased mature miR-7 production and attenuated inhibition of KLF4 translation. Mechanistically, enhanced oncogenic Yes associated protein (YAP) nuclear translocation mediates sequestration of p72, a co-factor of the Drosha/DGCR8 complex for pri-miR-7s processing, leading to attenuation of microprocessors' efficiency. Knockdown of YAP or transfection with a mature miR-7 mimic can significantly recover miR-7 expression to restore this feedback loop, and in turn to inhibit cancer cell growth by repressing KLF4 expression in vitro. Thus, our findings indicate that targeting the KLF4-miR-7 feedback loop might be a potential strategy for PCa therapy.

Elevated expression of Par3 promotes prostate cancer metastasis by forming a Par3/aPKC/KIBRA complex and inactivating the hippo pathway.

BACKGROUND: Prostate cancer (PCa) is one of the most frequent tumors and leading cause of cancer deaths among males worldwide. The majority of deaths are due to recurrence and subsequent development of the metastatic cancer. Although loss or dislocalization of polarity proteins has been implicated in embryogenesis deficiency and tumorigenesis, association of polarity protein expression levels with tumor metastasis remains unclear. METHODS: Bioinformatics, qRT-PCR, western blot and immunohistochemical (IHC) analyses were used to examine expression of Par3, a key component of polarity-associated partitioning defective (PAR) complex, in primary and metastatic clinical PCa samples. Loss-of-function and gain-of-function studies in vitro and in vivo were performed to determine the functions of Par3 during metastasis of PCa. Co-immunoprecipitation (co-IP), western blot, immunofluorescence (IF), chromatin immunoprecipitation (ChIP) and qRT-PCR analyses were conducted to investigate the underlying mechanism for the function of Par3 on PCa metastasis. RESULTS: In this study, we found that elevated expression of Par3 is positively associated with PCa metastasis. Knockdown of Par3 inhibits PCa cell migration and invasion in vitro and tumor metastasis in vivo, whereas overexpression of Par3 yields the opposite results. Mechanistically, Par3 suppresses phosphorylation of LATS to inactivate the Hippo pathway and enhances nuclear translocation of YAP by sequestrating KIBRA from the KIBRA/Merlin/FRMD6 complex and forming a Par3/aPKC/KIBRA complex. Stable knockdown of Par3 leads to restoration of the KIBRA/Merlin/FRMD6 complex and activation of the Hippo pathway, and then results in an inhibition on YAP nuclear translocation. In addition, in conjunction with the TEA domain (TEAD) transcription factor family, intranuclear YAP promotes the transcription of several pro-metastatic genes such as the matrix metalloproteinase (MMP) family, Zeb1, Snail1 and Twist1. Moreover, knockdown of Par3 downregulates expression of these pro-metastatic genes. CONCLUSIONS: Our findings indicate that elevated expression of Par3 promotes PCa metastasis via KIBRA sequestration-mediated inactivation of the Hippo pathway to upregulate expression of pro-metastatic genes. Downregulation of Par3 expression may serve as a potential treatment approach for PCa metastasis by activating the Hippo pathway.

MicroRNA-7 inhibits the stemness of prostate cancer stem-like cells and tumorigenesis by repressing KLF4/PI3K/Akt/p21 pathway.

Up to now, the molecular mechanisms underlying the stemness of prostate cancer stem cells (PCSCs) are still poorly understood. In this study, we demonstrated that microRNA-7 (miR-7) appears to be a novel tumor-suppressor miRNA, which abrogates the stemness of PCSCs and inhibits prostate tumorigenesis by suppressing a key stemness factor KLF4. MicroRNA-7 is down-regulated in prostate cancer cells compared to non-tumorigenic prostate epithelial cells. Restoration of miR-7 suppresses the expression of the stemness factor KLF4 in PCSCs and inhibits prostate tumorigenesis both in vitro and in vivo. Interestingly, the suppression of the stemness of PCSCs by miR-7 is sustained for generations in xenografts. Analysis of clinical samples also revealed a negative correlation between miR-7 expression and prostate tumor progression. Mechanistically, overexpression of miR-7 may lead to a cell cycle arrest but not apoptosis, which seems achieved via suppressing the KLF4/PI3K/Akt/p21 pathway. This study identifies miR-7 as a suppressor of PCSCs' stemness and implicates its potential application for PCa therapy.

MicroRNAs targeting prostate cancer stem cells.

Prostate cancer is a frequently diagnosed cancer in males with high mortality in the world. As a heterogeneous tissue, the tumor mass contains a subpopulation that is called as cancer stem cells and displays stem-like properties such as self-renewal, epithelial-mesenchymal transition, metastasis, and drug resistance. Cancer stem cells have been identified in variant tumors and shown to be regulated by various molecules including microRNAs. MicroRNAs are a class of small non-coding RNAs, which can influence tumorigenesis via different mechanisms. In this review, we focus on the functions of microRNAs on regulating the stemness of prostate cancer stem cells with different mechanisms and propose the potential roles of microRNAs in prostate cancer therapy.

Four microRNAs promote prostate cell proliferation with regulation of PTEN and its downstream signals in vitro.

BACKGROUND: Phosphatase and tensin homologue (PTEN), as a tumor suppressor, plays vital roles in tumorigenesis and progression of prostate cancer. However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs (miR-19b, miR-23b, miR-26a and miR-92a) promotes prostate cell proliferation by regulating PTEN and its downstream signals in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3' UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1. CONCLUSIONS: These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1 in vitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer.

Development of chimeric gene regulators for cancer-specific gene therapy with both transcriptional and translational targeting.

Cancer gene therapy has been of great challenge in achieving maximal high levels of specificity and more rational efficiency in target cancer cell. We herein developed a novel approach for cancer-specific gene therapy using both transcriptional and translational targeting regulation. We integrated the tumor-specific gene promoter of hTERT, the 5'UTR of bFGF-2, the enhancer of woodchuck hepatitis virus post-transcriptional regulatory element (WRE), and/or the 3'UTR of the human EGFR into two major chimeric gene regulators. We found that chimeric gene regulator I (hTERT_5'UTR...WRE_BGHpolyA) enhanced the specificity of expression in hepatocellular carcinoma (HCC) cells up to 300% in total due to increases at both the transcriptional and translational levels but only 120-200% enhancement at the transcriptional level and 120-180% enhancement at the translational level. In addition, chimeric gene regulator II (hTERT_5'UTR...WRE_3'UTR_BGHpolyA) improved the specificity to 550% and also highly strengthened the stability of the mRNA. In vitro cytotoxicity assays demonstrated that HCC cell growth was inhibited by HSV-1 TK expression under the control of both chimeric regulators, with a relative cell viability of approximately 80% for 2 days and approximately 85% for 4 days after transfection, respectively. These observations represent a new approach for highly tumor-specific gene expression and also provide insights into application to cancer gene therapy.

TAT-phiC31 integrase mediates DNA recombination in mammalian cells.

Streptomyces phage integrase phiC31 is capable of mediating site-specific insertions in mammalian genomes. To avoid potential toxicity of long-term expression of phiC31 in host cells, we developed a method employing a cell-permeable TAT-phiC31 integrase. His6-tagged phiC31 proteins with or without an HIV TAT intercellular transducing peptide were generated and purified. Both of them retained integrase activity in vitro. However, TAT-phiC31 but not phiC31 was able to mediate a specific integration between two att sites in the genome of 293-PB [EGFP] report cell line. Transduced TAT-phiC31 was mainly localized in the cytoplasm that is similar to the localization of phiC31 when expressed through cDNA transfection. Adding a nuclear localization signal (NLS) peptide to the C-terminus of TAT-phiC31 facilitated nuclear localization of the integrase with an increased efficiency of recombination in the reporter cell line. These results demonstrated that TAT can mediate a cell membrane entry of phiC31 protein to perform a site-specific integration in mammalian cells. This is a simple and possibly safer method of site-specific recombination for gene delivery.