Selective degradation of PL2L60 by metabolic stresses­induced autophagy suppresses multi­cancer growth.

It has been reported that PL2L60 proteins, a product of PIWIL2 gene which might be activated by an intragenic promoter, could mediate a common pathway specifically for tumorigenesis. In the present study, it was further identified by using western blot assay that the PL2L60 proteins could be degraded in cancer cells through a mechanism of selective autophagy in response to oxidative stress. The PL2L60 was downregulated in various types of cancer cells under the hypoxic condition independently of HIF­1α, resulting in apoptosis of cancer cells. Inhibition of autophagy by small interfering RNA targeting of either Beclin­1 (BECN1) or Atg5 resulted in restoration of PL2L60 expression in hypoxic cancer cell. The hypoxic degradation of PL2L60 was also blocked by the attenuation of the autophagosome membrane protein Atg8/microtubule­associated protein 1 light chain 3 (LC3) or autophagy cargo protein p62 expression. Surprisingly, Immunofluorescence analysis demonstrated that LC3 could be directly bound to PL2L60 and was required for the transport of PL2L60 from the nucleus to the cytoplasm for lysosomal flux under basal or activated autophagy in cancer cells. Moreover, flow cytometric analysis displayed that knocking down of PL2L60 mRNA but not PIWIL2 mRNA effectively inhibited cancer cell proliferation and promoted apoptosis of cancer cells. The similar results were obtained from in vivo tumorigenic experiment, in which PL2L60 downregulation in necroptosis areas was confirmed by immunohistochemistry. These results suggested that various cancer could be suppressed by promoting autophagy. The present study revealed a key role of autophagic degradation of PL2L60 in hypoxia­induced cancer cell death, which could be used as a novel therapeutic target of cancer.

A novel tumor-specific broad-spectral monoclonal antibody to PL2L60 is highly effective for the treatment of various types of cancers from human and mouse.

There are numerous antibodies used for cancer therapy in clinic, but they are essentially less efficacy than expected. None of them has tumor-specific and broad-spectral properties. PIWIL2-like (PL2L) protein 60 (PL2L60) is a product of alienated activation of PIWIL2 gene, and has been found to be specifically and widely expressed in various types of cancers, including hematopoietic and solid ones. Current study aims to investigate whether a monoclonal antibody (mAb) to PL2L60 has both tumor-specific and broad-spectral properties, which can be used universally to treat various types of cancers. The expression of PL2L60 protein in the cell surface and cytoplasm were determined in a panel of human and mouse tumor cell lines by flow cytometry, immunofluorescent microscopy and Western Blotting. The apoptosis and the cell cycle arrest of the tumor cells treated with mAb KAO3 were evaluated by flow cytometry. The tumorigenesis of the mAb KAO3-pretreated tumor cells was determined by tumor incidence and tumor size, and the efficacy of mAb KAO3 treatment on tumor growth in tumors-bearing mice were kinetically evaluated. Complement-dependent cytotoxicity (CDC) assay was used to determine the capacity of mAb KAO3 to kill tumor cells. Treatment of human or mouse tumor cells from hematopoietic or solid tumors with mAb KAO3 at the time of inoculation efficiently inhibited tumorigenesis in the severe combined immunodeficient (SCID) mice. Moreover, injection of mAb KAO3 into established tumors significantly inhibited their growth, and prolonged survival of the tumor-bearing mice, including lymphoma, breast cancer, lung cancer and cervical cancer. The efficacy of mAb KAO3 treatment is likely associated with its binding to PL2L60 expressed on tumor cell surface, which may lead to cancer cell death through blocking cell cycling and/or activation of complement. In conclusion, we have identified a tumor-specific mAb to PL2L60 (KAO3), which may be used potentially to treat all the types of human cancers including from both hematopoietic and solid ones.

Splenic irradiation combined with tumor irradiation promotes T cell infiltration in the tumor microenvironment and helps in tumor control.

Locally applied radiation to the tumor is reported to stimulate systemic immune response. During radiotherapy to the abdominal cancer, spleen often receives certain dose, though as an important immune organ, little is known about the impact of splenic irradiation (SI) on systemic immune and local tumor control. Through a mice model, we found that the combination of SI with tumor irradiation (TI) helped in local control. The analysis of the tumor infiltrating leucocytes demonstrated that SI plus TI brought more T cell aggregation in the tumor microenvironment (TME), which helped in tumor control. Increased T cell infiltration may be partly due to higher expression of T cell chemokine in the TME and more expression of CXCR3 on the T cells in the spleen after SI. SI produced more IL-1ß in the spleen, IL-1ß stimulated the expression of CXCR3 on the T cells, and enhanced their migration ability. Taken together, radiation to the spleen combined with TI helped in local control through promoting T cell infiltration, and may be a considerable means to enhance the immunomodulatory of radiotherapy.

Inhibiting the CD8+ T cell infiltration in the tumor microenvironment after radiotherapy is an important mechanism of radioresistance.

Endogenous immune response participates in tumor control, and radiotherapy has immune modulatory capacity, but the role of immune modulation in the tumor microenvironment invoked by radiotherapy in radiosensitivity is poorly defined. In the present study, a radio-resistant melanoma cell line was obtained after repeated irradiation to the parental tumor in C57BL/6 mice. Radiotherapy resulted in aggregation of CD8+ and CD3+ T cells, and decrease of myeloid-derived suppressor cells and dendritic cells in the parental tumor, but not in the resistant tumors. CD4+ T cells and B cells did not change significantly. The CD8+ T cell infiltration after radiotherapy is important for tumor response, because in the nude mice and CD8+ T cell-depleted C57BL/6 mice, the parental and resistant tumor has similar radiosensitivity. Patients with good radiation response had more CD8+ T cells aggregation after radiotherapy. Radiotherapy resulted in robust transcription of T cell chemoattractant in the parental cells, and the expression of CCL5 was much higher. These results reveal a novel mechanism of radioresistance, tumor cells inhibit the infiltration of CD8+ T cell after radiotherapy and become radioresistant. Increasing CD8+ T cell infiltration after RT may be an effective way to improve tumor radiosensitivity.

An unusual intragenic promoter of PIWIL2 contributes to aberrant activation of oncogenic PL2L60.

PIWIL2-like (PL2L) protein 60 (PL2L60), a product of aberrantly activated PIWIL2 gene, is widely expressed in various types of tumors and may promote tumorigenesis. However, the mechanisms underlying the activation of expression of PL2L60 remain unknown. In this study, an intragenic promoter responsible for the activation of PL2L60 within the human PIWIL2 gene has been identified, cloned and characterized. The promoter of PL2L60 is located in the intron 10 of the host gene PIWIL2. Bioinformatic and mutagenic analysis reveals that this intragenic promoter within the sequence of 50 nucleotides contains two closely arranged cis-acting elements specific for the hepatic leukemia factor (HLF) in the positive strand and signal transducer and activator of transcription 3 (STAT3) in the negative strand. Chromatin immunoprecipitation analysis demonstrates that both the HLF and polymerase II (Pol II), a hallmark of active promoters, directly bind to the sequence, although STAT3 does not. Knockdown of HLF and STAT3 alone or both by RNA interference significantly reduced both promoter activity and the PL2L60 protein expression, although there is no additive effect. The expression of PL2L60 proteins was enhanced when host gene Piwil2 was genetically disrupted in a murine cell model. Taken together, we have identified a PL2L60-specific intragenic promoter in the host gene of PIWIL2, which is interdependently activated by HLF and STAT3 through steric interaction. This activation is dependent on cellular milieu rather than the integrity of host gene PIWIL2, highlighting a novel, important mechanism for a cancer-causing gene to be activated during tumorigenesis.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1.

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

Adaptive and Acquired Resistance to EGFR Inhibitors Converge on the MAPK Pathway.

Both adaptive and acquired resistance significantly limits the efficacy of the epidermal growth factor receptor (EGFR) kinase inhibitors. However, the distinct or common mechanisms of adaptive and acquired resistance have not been fully characterized. Here, through systematic modeling of erlotinib resistance in lung cancer, we found that feedback reactivation of MAPK signaling following erlotinib treatment, which was dependent on the MET receptor, contributed to the adaptive resistance of EGFR inhibitors. Interestingly, acquired resistance to erlotinib was also associated with the MAPK pathway activation as a result of CRAF or NRAS amplification. Consequently, combined inhibition of EGFR and MAPK impeded the development of both adaptive and acquired resistance. These observations demonstrate that adaptive and acquired resistance to EGFR inhibitors can converge on the same pathway and credential cotargeting EGFR and MAPK as a promising therapeutic approach in EGFR mutant tumors.

An asymmetrically dimethylarginated nuclear 90 kDa protein (p90aDMA) induced by interleukin (IL)-2, IL-4 or IL-6 in the tumor microenvironment is selectively degraded by autophagy.

Protein arginine methylation is a common posttranslational modification resulting in the generation of asymmetric dimethylarginine (aDMA) and symmetric dimethylarginine (sDMA). Currently, the regulation of aDMA or sDMA by hypoxia, nutrient stavation or cytokines in the tumor microenvironment remains largely unknown. Here we show that p90aDMA, p70aDMA and p90sDMA, endogenous proteins containing aDMA or sDMA with mass 70 or 90 kDa, were widely and dominantly expressed in breast cancer cell lines. Notably, it was p90aDMA rather than p90sDMA that accumulated in the nucleus upon stimulation of cancer cells with interleukin (IL)-2, IL-4, IL-6 but not IL-8. In addition, the p90aDMA accumulation could be inhibited after treatment with a global methyltrasferase inhibitor, adenosine-2',3'-dialdehyde (AdOx). It seemed that some endogenous proteins in cancer cells were asymmetrically arginine-methylated upon exposure to some cytokines.. Furthermore, endogenous proteins of aDMA, such as p90aDMA and p70aDMA, were degraded in response to hypoxia, nutrient starvation and rapamycin treatment in breast and cervical cancer cells. IL-2/4/6 slightly increased basal autophagy but slightly decreased the rapamycin­induced autophagy in cancer cells, suggesting that IL-2/4/6 and autophagy inducers play distinct roles in the regulation of aDMA of proteins. Conversely, rapamycin accumulated p90sDMA in MDA-MB­231 and MCF-7 cells. Taken together, our results add a new dimension to the complexity of arginine methylated regulation in response to various stimuli and provide the first evidence that aDMA serves as one specific degradation signal of selective autophagy.

Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-ß2 by targeting ZNF217 via miR-200c in keloid fibroblasts.

Abnormally high activation of transforming growth factor-ß (TGF-ß) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-ß (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-ß in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-ß, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-ß2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-ß2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-ß2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-ß2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

MicroRNA-101 targets von Hippel-Lindau tumor suppressor (VHL) to induce HIF1α mediated apoptosis and cell cycle arrest in normoxia condition.

The activation/inactivation of HIF1α is precisely regulated in an oxygen-dependent manner. HIF1α is essential for hypoxia induced apoptosis and cell cycle arrest. Several recent studies indicated that the expression of miRNAs can be modulated by hypoxia. However, the involvement of miRNAs in the regulation of HIF1α induction remains elusive. In present study, we demonstrated that miR-101 was rapidly and transiently induced after hypoxia in breast cancer cells. Over-expression of miR-101 significantly inhibited cell proliferation in breast cancer cells through increased apoptosis and cell cycle arrest in normoxia condition. This inhibitory phenomenon seems due to miR-101-mediated induction of HIF1α, because we identified that VHL, a negative regulator of HIF1α, is a novel target of miR-101 and over-expression of miR-101 decreased VHL levels and subsequently stabilized HIF1α and induced its downstream target VEGFA. Furthermore, we demonstrated that siRNA-mediated knockdown of VHL or HIF1α overexpression could also induce apoptosis and cell cycle arrest whereas enforced expression of VHL, administration of anti-miR-101 oligos or treatment of 2-MeOE2, an inhibitor of HIF1α, could rescue cells from such inhibition. These results reveal a novel regulatory mechanism of HIF1α induction in normoxia and suggest that miR-101 mediated proliferation inhibition may through HIF1α mediated apoptosis and cell cycle arrest.

Beclin-1-independent autophagy mediates programmed cancer cell death through interplays with endoplasmic reticulum and/or mitochondria in colbat chloride-induced hypoxia.

Autophagy has dual functions in cell survival and death. However, the effects of autophagy on cancer cell survival or death remain controversial. In this study, we show that Autophagy can mediate programmed cell death (PCD) of cancer cells in responding to cobalt chloride (CoCl2)-induced hypoxia in a Beclin-1-independent but autophagy protein 5 (ATG5)-dependent manner. Although ATG5 is not directly induced by CoCl2, its constitutive expression is essential for CoCl2-induced PCD. The ATG5-mediated autophagic PCD requires interplays with endoplasmic reticulum (ER) and/or mitochondria. In this process, ATG5 plays a central role in regulating ER stress protein CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and mitochondrial protein second mitochondria derived activator of caspases (Smac). Two pathways for autophagic PCD in cancer cells responding to hypoxia have been identified: ATG5/CHOP/Smac pathway and ATG5/Smac pathway, which are probably dependent on the context of cell lines. The former is more potent than the latter for the induction of PCD at the early stage of hypoxia, although the ultimate efficiency of both pathways is comparable. In addition, both pathways may require ATG5-mediated conversion of LC3-I into LC3-II. Therefore, we have defined two autophagy-mediated pathways for the PCD of cancer cells in hypoxia, which are dependent on ATG5, interplayed with ER and mitochondria and tightly regulated by hypoxic status. The findings provide a new evidence that autophagy may inhibit tumor cell proliferation through trigger of PCD, facilitating the development of novel anti-cancer drugs.

Metformin can block precancerous progression to invasive tumors of bladder through inhibiting STAT3-mediated signaling pathways.

BACKGROUND: Metformin is the first line of oral antidiabetic drug in the biguanide class for treatment of type 2 diabetes. Increasing evidence has suggested that it is a potential anti-tumor drug. However, the mechanisms underlying inhibiting tumor development remain elusive, especially in bladder tumors. METHODS: T24 and J82 cell lines were used as an in vitro model, and 24 female SD rats were used to build an N-methyl-N-nitrosourea (MNU)-induced orthotopic rat bladder cancer model. Transfection of lentivirus-based shRNA was used to construct the STAT3-KNOCKDOWN T24 cell line. After metformin treatment, the viability of bladde cancer cells was determined by CCK8. Cell cycle distribution and apoptosis were assessed by flow cytometry. The migration and invasion abilities of cells were evaluated by wound healing and transwell asssays. The inactivation of stat3 pahtway was examined by qRTPCR, western blot and Immunofluorescence. RESULTS: Metformin can effectively inhibit precancerous progression to invasive cancer in an MNU-induced rat orthotopic bladder tumor model, although it could not completely suppress normal cells transforming into tumor cells. While the MNU could induce 50 % rats (4/8) to develop invasive bladder cancers, the rats co-administrated with metformin failed to develop invasive tumors but retained at precancerous or non-invasive stages, exhibiting as dysplasia, papillary tumor and/or carcinoma in situ (CIS). Accordingly, phosphorylation of signal transducer and activator of transcription 3 (STAT3), which is a well known oncogene, was significantly inhibited in the tumors of rats treated with metformin. In vitro experiments revealed that the metformin could efficiently inhibit STAT3 activation, which was associated with the cell cycle arrest, reduction of cell proliferation, migration and invasiveness, and increase in apoptotic cell death of bladder cancer cell lines. CONCLUSIONS: These findings provide for the first time the evidence that metformin can block precancerous lesions progressing to invasive tumors through inhibiting the activation of STAT3 pathway, and may be used for treatment of the non-invasive bladder cancers to prevent them from progression to invasive tumors.

A requirement of dendritic cell-derived interleukin-27 for the tumor infiltration of regulatory T cells.

Tregs (Foxp3+CD4+) are enriched in tumors to foster a tolerant microenvironment that inhibits antitumor immune response. IL-27 is reported to regulate the development and function of Tregs in vitro and in vivo; however, the effects of endogenous IL-27 on Tregs in the tumor microenvironment remain elusive. We demonstrated that in the absence of DC-derived IL-27, Tregs were decreased significantly in transplanted B16 melanoma, transplanted EL-4 lymphoma, and MCA-induced fibrosarcoma by using IL-27p28 conditional KO mice. Further studies revealed that IL-27 promoted the expression of CCL22, which is established to mediate the recruitment of peripheral Tregs into tumors. Tumor-associated DCs were identified as the major source of CCL22 in tumor sites, and IL-27 could induce CCL22 expression in an IL-27R-dependent manner. Intratumoral reconstitution of rmCCL22 or rmIL-27, but not rmIL-27p28, significantly restored the tumor infiltration of Tregs in IL-27p28 KO mice. Correlated with a decreased number of Tregs, tumor-infiltrating CD4 T cells were found to produce much more IFN-γ in IL-27p28 KO mice, which highlighted the physiological importance of Tregs in suppressing an antitumor immune response. Overall, our results identified a novel mechanism of action of IL-27 on Tregs in the context of cancers.

Critical role of dendritic cell-derived IL-27 in antitumor immunity through regulating the recruitment and activation of NK and NKT cells.

Critical roles of IL-27 in autoimmune diseases and infections have been reported; however, the contribution of endogenous IL-27 to tumor progression remains elusive. In this study, by using IL-27p28 conditional knockout mice, we demonstrate that IL-27 is critical in protective immune response against methyl-cholanthrene-induced fibrosarcoma and transplanted B16 melanoma, and dendritic cells (DCs) are the primary source. DC-derived IL-27 is required for shaping tumor microenvironment by inducing CXCL-10 expression in myeloid-derived suppressor cells and regulating IL-12 production from DCs, which lead to the recruitment and activation of NK and NKT cells resulting in immunological control of tumors. Indeed, reconstitution of IL-27 or CXCL-10 in tumor site significantly inhibits tumor growth and restores the number and activation of NK and NKT cells. In summary, our study identifies a previous unknown critical role of DC-derived IL-27 in NK and NKT cell-dependent antitumor immunity through shaping tumor microenvironment, and sheds light on developing novel therapeutic approaches based on IL-27.

Early to intermediate steps of tumor embolic formation involve specific proteolytic processing of E-cadherin regulated by Rab7.

The lymphovascular embolus is an enigmatic entity adept at metastatic dissemination and chemotherapy resistance. Using MARY-X, a human breast cancer xenograft that exhibits florid lymphovascular emboli in mice and spheroids in vitro, we established a model where the in vitro transition stages from minced tumoral aggregates to well-formed spheroids served as a surrogate for in vivo emboli formation. MARY-X well-formed spheroids and emboli exhibited strong similarity of expression. The aggregate-to-spheroid transition stages were characterized by increased ExoC5, decreased Hgs and Rab7, increased calpains, increased full-length E-cadherin (E-cad/FL), and the transient appearance of E-cad/NTF2, a 95 kDa E-cadherin fragment and increased Notch3icd (N3icd), the latter two fragments produced by increased γ-secretase. Both transient and permanent knockdowns of Rab7 in MCF-7 cells increased protein but not transcription of E-cad/FL and resulted in the de novo appearance of E-cad/NTF2, the presence of nuclear E-cad/CTF2, and increased Notch1icd (N1icd). Overexpression of Rab7 conversely decreased E-cad/FL, γ-secretase (PS1/NTF), and E-cad/NTF2. Overexpression of calpains did not alter PS1/NTF but decreased E-cad/FL and E-cad/NTF2 and increased N1icd. Well-formed spheroids showed increased Rab7, absent E-cad/NTF2, decreased PS1/NTF, increased E-cad/NTF1, and increased N3icd, the latter two fragments being the direct and indirect consequences, respectively, of increased calpains (calpain 1 and calpain 2). Inhibition of calpains decreased E-cad/NTF1 but increased E-cad/NTF2 showing that calpains compete with γ-secretase (PS1) for closely located cleavage/binding sites on E-cadherin and that increased calpains can shuttle even decreased levels of γ-secretase to Notch 3, resulting in increased Notch 3 signaling in the well-formed spheroids.

Singular v dual inhibition of SNF2L and its isoform, SNF2LT, have similar effects on DNA damage but opposite effects on the DNA damage response, cancer cell growth arrest and apoptosis.

SNF2L, an ATPase chromatin remodeling gene nearly ubiquitously expressed in diverse tissues, cancers, and derived cell lines, contributes to the chromatin remodeling complex that facilitates transcription. Because of this near ubiquitous expression, it has not been exploited as a cancer therapeutic target. However, in a recent study, we found that highly malignant cancer cells, although expressing SNF2L at similar levels as their normal counterparts, were sensitive to its knockdown. Only the highly malignant (HM) lines showed significant growth inhibition, DNA damage, a DNA damage response, and phosphorylation of checkpoint proteins and marked apoptosis. In studying SNF2L, we discovered a novel truncated isoform, SNF2LT which, when compared to full length SNF2L, lacked three important domains: HAND, SANT and SLIDE. Although truncated isoforms usually have antagonistic functions to their parental molecule, here SNF2LT knockdown had similar effects to the knockdown of its parental molecule, SNF2L, of inducing DNA damage, a DNA damage response, cell cycle arrest and apoptosis selectively in cancer cell lines. However dual SNF2L and SNF2LT knockdown, while inducing DNA damage, did not result in a DNA damage response, a cell cycle arrest and apoptosis. In fact HM lines subjected to dual knockdown paradoxically exhibited sustained cell growth. Our findings indicate that the ratio of SNF2L to its isoform tightly regulates the cancer cell's response to DNA damage. Cancer cell lines which endogenously express low levels of both SNF2L and its isoform mimic the situation of dual knockdown and permit DNA damage which is allowed to propagate unchecked.

Germline stem cell gene PIWIL2 mediates DNA repair through relaxation of chromatin.

DNA damage response (DDR) is an intrinsic barrier of cell to tumorigenesis initiated by genotoxic agents. However, the mechanisms underlying the DDR are not completely understood despite of extensive investigation. Recently, we have reported that ectopic expression of germline stem cell gene PIWIL2 is associated with tumor stem cell development, although the underlying mechanisms are largely unknown. Here we show that PIWIL2 is required for the repair of DNA-damage induced by various types of genotoxic agents. Upon ultraviolet (UV) irradiation, silenced PIWIL2 gene in normal human fibroblasts was transiently activated after treatment with UV light. This activation was associated with DNA repair, because Piwil2-deficienct mouse embryonic fibroblasts (mili(-/-) MEFs) were defective in cyclobutane pyrimidine dimers (CPD) repair after UV treatment. As a result, the UV-treated mili(-/-) MEFs were more susceptible to apoptosis, as characterized by increased levels of DNA damage-associated apoptotic proteins, such as active caspase-3, cleaved Poly (ADP-ribose) polymerase (PARP) and Bik. The impaired DNA repair in the mili(-/-) MEFs was associated with the reductions of histone H3 acetylation and chromatin relaxation, although the DDR pathway downstream chromatin relaxation appeared not to be directly affected by Piwil2. Moreover, guanine-guanine (Pt-[GG]) and double strand break (DSB) repair were also defective in the mili(-/-) MEFs treated by genotoxic chemicals Cisplatin and ionizing radiation (IR), respectively. The results indicate that Piwil2 can mediate DNA repair through an axis of Piwil2 → histone acetylation → chromatin relaxation upstream DDR pathways. The findings reveal a new role for Piwil2 in DNA repair and suggest that Piwil2 may act as a gatekeeper against DNA damage-mediated tumorigenesis.

Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Homozygous deletion but not mutation of exons 5 and 8 of the fragile histidine triad (FHIT) gene is associated with features of differentiated thyroid carcinoma.

The fragile histidine triad (FHIT) gene encompasses the most common human fragile site, FRA3B at 3p14.2, a region that is involved in homozygous deletions in a variety of human tumors. FHIT is considered to be a tumor suppressor gene that is frequently inactivated in various types of cancer. To study the role of the FHIT gene in thyroid tumorigenesis, we looked for homozygous deletions or mutations of exons 5 and 8 of the FHIT gene in 65 cases of differentiated thyroid carcinoma (DTC) and their matched non-cancerous epithelium (NCE), using exon-specific PCR amplification and PCR single strand conformation polymorphism (PCR-SSCP) techniques. In DTC, the incidence of homozygous deletion of exon 5 was 30.8% (20/65), and it was associated with tumor metastasis to lymph nodes (p <0.05). The incidence of homozygous deletion of exon 8 was 29.2% (19/65), and it was associated with the tumor pathological grade, TNM stage, and lymph node metastasis (p <0.05). There was strong correlation between homozygous deletions of exon 5 and exon 8 (p <0.01). No point mutations were observed in either exon 5 or exon 8. These findings suggest that: (a) exons 5 and 8 of FHIT are key target regions of deletion, (b) homozygous deletions of exon 5 and exon 8 may be good biomarkers for the biological behavior of DTC, and (c) point mutation of these exons may not be involved in the inactivation of the FHIT gene in DTC.

Association of the promoter methylation and protein expression of Fragile Histidine Triad (FHIT) gene with the progression of differentiated thyroid carcinoma.

The role of aberrant methylation of fragile histidine triad (FHIT) promoters in the differentiated thyroid carcinoma (DTC) is not yet clear. Therefore, we investigated the association of the status of FHIT promoter methylation and FHIT protein expression with the clinicopathological progression of DTC, using PCR-based methylation assay and immunohistochemical technique. While no FHIT gene promoter methylation was observed in the matched non-cancerous epithelium (NCE) specimens, 24.6% of DTC samples demonstrated methylation in the FHIT promoter region. The protein expression of FHIT in NCE and DTC was 100.0% and 41.5% (P<0.01), respectively. There was a negative correlation between promoter methylation and protein expression of FHIT gene (P<0.05). Additionally, the methylation status appeared to be significantly associated with the pathological grade, tumor TNM stage, and lymph node metastasis (P<0.05), and FHIT proteins were weakly expressed in only about 20% of DTC with grade II pathological changes, TNM stage III/IV, or lymph node metastasis. Finally, the gender and tumor classification but not age marginally affected the promoter methylation and protein expression of FHIT. Our results suggest that methylation of the promoter region may play a key role in inactivation of FHIT - possibly leading to subsequent carcinogenesis and progression of DTC.