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Piezo2 mechanotransduction channel is a crucial mediator of sensory neurons for sensing and transducing touch, vibration, and proprioception. We here characterized Piezo2 expression and cell specificity in rat peripheral sensory pathway using a validated Piezo2 antibody. Immunohistochemistry using this antibody revealed Piezo2 expression in pan primary sensory neurons of dorsal root ganglia in naive rats, which was actively transported along afferent axons to both central presynaptic terminals innervating the spinal dorsal horn (DH) and peripheral afferent terminals in the skin. Piezo2 immunoreactivity (IR) was also detected in the postsynaptic neurons of the DH and in the motor neurons of the ventral horn, but not in spinal glial fibrillary acidic protein-positive and Iba1-positive glia. Notably, Piezo2-IR was clearly identified in peripheral nonneuronal cells, including perineuronal glia, Schwann cells in the sciatic nerve and surrounding cutaneous afferent endings, as well as in skin epidermal Merkel cells and melanocytes. Immunoblots showed increased Piezo2 in dorsal root ganglia ipsilateral to plantar injection of complete Freund's adjuvant, and immunostaining revealed increased Piezo2-IR intensity in the DH ipsilateral to complete Freund's adjuvant injection. This elevation of DH Piezo2-IR was also evident in various neuropathic pain models and monosodium iodoacetate knee osteoarthritis pain model, compared with controls. We conclude that (1) the pan neuronal profile of Piezo2 expression suggests that Piezo2 may function extend beyond simply touch or proprioception mediated by large-sized low-threshold mechanosensitive primary sensory neurons; (2) Piezo2 may have functional roles involving sensory processing in the spinal cord, Schwann cells, and skin melanocytes; and (3) aberrant Piezo2 expression may contribute pain pathogenesis.

The Sigma-1 receptor (σ(1)R) is highly expressed in the primary sensory neurons (PSNs) that are the critical site of initiation and maintenance of pain following peripheral nerve injury. By immunoblot and immunohistochemistry, we observed increased expression of both σ(1)R and σ1R-binding immunoglobulin protein (BiP) in the lumbar (L) dorsal root ganglia (DRG) ipsilateral to painful neuropathy induced by spared nerve injury (SNI). To evaluate the therapeutic potential of PSN-targeted σ(1)R inhibition at a selected segmental level, we designed a recombinant adeno-associated viral (AAV) vector expressing a small hairpin RNA (shRNA) against rat σ(1)R. Injection of this vector into the L4/L5 DRGs induced downregulation of σ(1)R in DRG neurons of all size groups, while expression of BiP was not affected. This was accompanied by attenuation of SNI-induced cutaneous mechanical and thermal hypersensitivity. Whole-cell current-clamp recordings of dissociated neurons showed that knockdown of σ(1)R suppressed neuronal excitability, suggesting that σ(1)R silencing attenuates pain by reversal of injury-induced neuronal hyperexcitability. These findings support a critical role of σ(1)R in modulating PSN nociceptive functions, and that the nerve injury-induced elevated σ(1)R activity in the PSNs can be a significant driver of neuropathic pain. Further understanding the role of PSN-σ(1)R in pain pathology may open routes to exploit this system for DRG-targeted pain therapy.

The duplication and deletion mutations of the S-SCAM/MAGI-2 gene are associated with schizophrenia and infantile spasms, respectively. S-SCAM is a unique synaptic scaffolding protein that localizes to both excitatory and GABAergic synapses. However, consequences of aberrant S-SCAM expression on GABAergic synapses is little studied. Here we report the effect of S-SCAM knockdown and overexpression on GABAergic synapses. S-SCAM knockdown in cultured hippocampal neurons caused a drastic loss of both pre- and post-synaptic components of GABAergic synapses, indicating its essential role in GABAergic synapse formation and maintenance. Surprisingly, S-SCAM overexpression also attenuated GABAergic synapses, but the effect is mediated by the loss of postsynaptic GABAA receptors, gephyrin, and neuroligin 2 and does not involve presynaptic component vesicular GABA transporters. Overexpression studies using S-SCAM mutants with various domain deletions indicated that GABAergic synapse loss correlates with their ability to increase excitatory synaptic function. Consistently, AMPA receptor antagonist CNQX or calcineurin inhibitor FK506 abolished the S-SCAM overexpression-induced loss of GABAA receptors, supporting that GABAergic synapse loss by S-SCAM overexpression is due to the activity-induced dispersal of synaptic GABAA receptors. These results suggest that abnormal S-SCAM protein levels disrupt excitation/inhibition balance in neurons, which may explain the pathogenic nature of S-SCAM copy number variations.

Oncogenic Ras mutants, and most frequently KRas mutants (86% of Ras-driven cancers), are found in approximately 25% of human cancers and are high-priority anticancer drug targets. Despite 30 years of effort to develop drugs that directly target oncogenic Ras mutants, no effective pharmacologic inhibitors for these mutants are clinically available, mainly because of the lack of suitable surface binding pockets for small molecules. More than 50 therapeutic antibodies have been clinically approved against many extracellular proteins. However, such antibodies do not have the capacity to localize in intracellular cytosolic regions after receptor-mediated endocytosis, restricting their therapeutic application for targeting cytosolic proteins. Our group recently developed a platform technology of cytosol-penetrating antibody, which in the IgG format can reach the cytosolic space of living cells owing to its endosomal escaping ability after receptor-mediated endocytosis. Exploiting the cytosol-penetrating antibody technology, we have engineered a human IgG1 format antibody, named iMab (internalizing and protein-protein interaction [PPI] interfering monoclonal antibody), which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of oncogenic Ras mutants. iMab specifically binds to the PPI interfaces of activated Ras with effector proteins to block the associations, thereby inhibiting the Ras downstream oncogenic signaling and exerting antiproliferation effects on oncogenic Ras mutant tumor cells. For in vivo antitumor efficacy assessment, we further engineer iMab to have tumor tissue-homing ability by fusion of tumor-associated integrin αvβ3/αvβ5 binding cyclic peptide to the N-terminus of light chain. When systemically administered, the iMab variant significantly inhibited the in vivo growth of oncogenic Ras-mutated tumor xenografts in mice, but not wild-type Ras-harboring tumors. Our results demonstrate the feasibility of developing antibody therapeutics that directly target cytosolic proteins involved in disease-associated PPIs, such as oncogenic Ras mutants, by systemic administration, similar to conventional therapeutic antibody regimens. Because the oncogenic Ras targeting antibody holds many desirable features of the conventional IgG antibody, it shows great potential for development as a first-in-class anticancer antibody. Citation Format: Seung-Min Shin, Ji-Sun Kim, Jin-Sun Hong, Seong-wook Park, Sei-Yong Jun, Hye-Jin Kweon, Dong-Ki Choi, Yong-Sung Kim. Direct targeting oncogenic Ras mutants by IgG-format cytosol-penetrating antibody [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B28.

The main obstacles for practical uses of cytosol-penetrating peptides and proteins include their lack of cell- or tissue-specific targeting and limited cytosolic access owing to the poor endosomal escape ability. We have previously reported a cytosol-penetrating, human IgG1 antibody TMab4-WYW, generally referred to as a cytotransmab (CT), which reaches the cytosol of living cells but nonspecifically because it is endocytosed via a ubiquitously expressed receptor called heparan sulfate proteoglycan (HSPG). Here, our aim was to construct a next-generation CT with tumor cell specificity and improved endosomal escape efficiency. We first substantially reduced the HSPG-binding activity of TMab4-WYW and then fused a cyclic peptide specifically recognizing tumor-associated epithelial cell adhesion molecule (EpCAM) to the N terminus of the light chain for EpCAM-mediated endocytosis, while maintaining the endosomal escape ability in the light chain variable domain (VL), thus generating epCT05. Then, we separately engineered another CT, dubbed epCT65-AAA, with an endosomal escape ability only in the heavy chain variable domain (VH) but not in VL, by functional grafting of the endosomal escape motif of epCT05 VL to the VH. We finally combined the heavy chain of epCT65-AAA and the light chain of epCT05 to create epCT65 with endosomal escape capacity in both the VH and VL. epCT65 effectively localized to the cytosol of only EpCAM-expressing tumor cells and showed approximately twofold improved endosomal escape efficiency, as compared with CTs with endosomal escape motifs in either VH or VL. The full-IgG format CT, epCT65, with a tumor cell-specific cytosol-penetrating activity, has a great potential for practical medical applications, e.g., as a carrier for cytosolic delivery of payloads.

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

Accumulating genetic evidence suggests that schizophrenia (SZ) is associated with individually rare copy number variations (CNVs) of diverse genes, often specific to single cases. However, the causality of these rare mutations remains unknown. One of the rare CNVs found in SZ cohorts is the duplication ofSynaptic Scaffolding Molecule(S-SCAM, also calledMAGI-2), which encodes a postsynaptic scaffolding protein controlling synaptic AMPA receptor levels, and thus the strength of excitatory synaptic transmission. Here we report that, in a transgenic mouse model simulating the duplication conditions, elevation of S-SCAM levels in excitatory neurons of the forebrain was sufficient to induce multiple SZ-related endophenotypes. S-SCAM transgenic mice showed an increased number of lateral ventricles and a reduced number of parvalbumin-stained neurons. In addition, the mice exhibited SZ-like behavioral abnormalities, including hyperlocomotor activity, deficits in prepulse inhibition, increased anxiety, impaired social interaction, and working memory deficit. Notably, the S-SCAM transgenic mice showed a unique sex difference in showing these behavioral symptoms, which is reminiscent of human conditions. These behavioral abnormalities were accompanied by hyperglutamatergic function associated with increased synaptic AMPA receptor levels and impaired long-term potentiation. Importantly, reducing glutamate release by the group 2 metabotropic glutamate receptor agonist LY379268 ameliorated the working memory deficits in the transgenic mice, suggesting that hyperglutamatergic function underlies the cognitive functional deficits. Together, these results contribute to validate a causal relationship of the rare S-SCAM CNV and provide supporting evidence for the rare CNV hypothesis in SZ pathogenesis. Furthermore, the S-SCAM transgenic mice provide a valuable new animal model for studying SZ pathogenesis.

Recombinant adeno-associated viral (AAV)-mediated therapeutic gene transfer to dorsal root ganglia (DRG) is an effective and safe tool for treating chronic pain. However, AAV with various constitutively active promoters leads to transgene expression predominantly to neurons, while glial cells are refractory to AAV transduction in the peripheral nervous system. The present study evaluated whether in vivo satellite glial cell (SGC) transduction in the DRG can be enhanced by the SGC-specific GFAP promoter and by using shH10 and shH19, which are engineered capsid variants with Muller glia-prone transduction. Titer-matched AAV6 (as control), AAVshH10, and AAVshH19, all encoding the EGFP driven by the constitutively active CMV promoter, as well as AAV6-EGFP and AAVshH10-EGFP driven by a GFAP promoter (AAV6-GFAP-EGFP and AAVshH10-GFAP-EGFP), were injected into DRG of adult male rats. Neurotropism of gene expression was determined and compared by immunohistochemistry. Results showed that injection of AAV6- and AAVshH10-GFAP-EGFP induces robust EGFP expression selectively in SGCs, whereas injection of either AAVshH10-CMV-EGFP or AAVshH19-CMV-EGFP into DRG resulted in a similar in vivo transduction profile to AAV6-CMV-EGFP, all showing efficient transduction of sensory neurons without significant transduction of glial cell populations. Coinjection of AAV6-CMV-mCherry and AAV6-GFAP-EGFP induces transgene expression in neurons and SGCs separately. This report, together with our prior studies, demonstrates that the GFAP promoter rather than capsid tropism determines selective gene expression in SGCs following intraganglionic AAV delivery in adult rats. A dual AAV system, one with GFAP promoter and the other with CMV promoter, can efficiently express transgenes selectively in neurons versus SGCs.

Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology., Oncogenic RAS mutants are key anti-cancer targets as KRas mutations are very frequent in human cancers. Here, the authors engineer a cytosol-penetrating anti-Ras antibody and demonstrate its ability to block RAS-effector protein interactions inhibiting tumour growth of Ras mutant-driven cancers.

How does chronic activity modulation lead to global remodeling of proteins at synapses and synaptic scaling? Here we report a role of guanylate-kinase-associated-protein (GKAP; also known as SAPAP), a scaffolding molecule linking NMDA receptor-PSD-95 to Shank-Homer complexes, in these processes. Over-excitation removes GKAP from synapses via ubiquitin-proteasome system, while inactivity induces synaptic accumulation of GKAP in rat hippocampal neurons. The bi-directional changes of synaptic GKAP levels are controlled by specific CaMKII isoforms coupled to different Ca2+ channels. α-CaMKII activated by NMDA receptor phosphorylates Serine-54 of GKAP to induce poly-ubiquitination of GKAP. In contrast, β-CaMKII activation via L-type voltage-dependent calcium channel promotes GKAP recruitment by phosphorylating Serine-340 and Serine-384 residues, which uncouples GKAP from MyoVa motor complex. Remarkably, overexpressing GKAP turnover mutants not only hampers activity-dependent remodeling of PSD-95 and Shank but also blocks bi-directional synaptic scaling. Therefore, activity-dependent turnover of PSD proteins orchestrated by GKAP is critical for homeostatic plasticity.

Human cervical cancer oncogene 1, HCCR-1, is over-expressed in various human tumors and appears to serve as a negative regulator of the p53 gene. HCCR-1 transgenic mice developed breast cancers but it is unknown how HCCR-1 contributes to tumorigenesis. We identified the HCCR-1 binding protein 3 (HCCRBP-3) as a binding partner for HCCR-1. These two proteins co-localized to the mitochondria. HCCRBP-3 over-expressed in various human tumors converted normal cells into tumor cells in vitro. Nude mice injected with NIH/3T3 cells stably transfected with HCCRBP-3 also induced the tumor formation. In addition, p53 showed the functional impairment in HCCRBP-3-transfected cells as accompanied with defective induction of p21 and bax. In support of this, p21 promoter activities containing p53 responsive elements were inhibited by HCCRBP-3 in a dose-dependent manner. Therefore, our study suggests that HCCRBP-3 contributes to the HCCR-1 induced tumorigenesis by interrupting the p53 function.

Synaptic plasticity, the cellular basis of learning and memory, involves the dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses. One of the remaining key unanswered aspects of AMPAR trafficking is the mechanism by which synaptic strength is preserved despite protein turnover. In particular, the identity of AMPAR scaffolding molecule(s) involved in the maintenance of GluA2-containing AMPARs is completely unknown. Here we report that the synaptic scaffolding molecule (S-SCAM; also called membrane-associated guanylate kinase inverted-2 and atrophin interacting protein-1) plays the critical role of maintaining synaptic strength. Increasing S-SCAM levels in rat hippocampal neurons led to specific increases in the surface AMPAR levels, enhanced AMPAR-mediated synaptic transmission, and enlargement of dendritic spines, without significantly effecting GluN levels or NMDA receptor (NMDAR) EPSC. Conversely, decreasing S-SCAM levels by RNA interference-mediated knockdown caused the loss of synaptic AMPARs, which was followed by a severe reduction in the dendritic spine density. Importantly, S-SCAM regulated synaptic AMPAR levels in a manner, dependent on GluA2 not GluA1, sensitive toN-ethylmaleimide-sensitive fusion protein interaction, and independent of activity. Further, S-SCAM increased surface AMPAR levels in the absence of PSD-95, while PSD-95 was dependent on S-SCAM to increase surface AMPAR levels. Finally, S-SCAM overexpression hampered NMDA-induced internalization of AMPARs and prevented the induction of long term-depression, while S-SCAM knockdown did not. Together, these results suggest that S-SCAM is an essential AMPAR scaffolding molecule for the GluA2-containing pool of AMPARs, which are involved in the constitutive pathway of maintaining synaptic strength.

Obese women have an increased risk for post-menopausal breast cancer. The physiological mechanism by which obesity contributes to breast tumourigenesis is not understood. We previously showed that HCCR-1 oncogene contributes to breast tumourigenesis as a negative regulator of p53 and detection of HCCR-1 serological level was useful for the diagnosis of breast cancer(.) In this study, we found that the HCCR-1 level is elevated in breast cancer tissues and cell lines compared to normal breast tissues. We identified apolipoprotein E (ApoE) interacting with HCCR-1. Our data show that HCCR-1 inhibits anti-proliferative effect of ApoE, which was mediated by diminishing ApoE secretion of breast cancer cells. Finally, HCCR-1 induced the severe obesity in transgenic mice. Those obese mice showed severe hyperlipidaemia. In conclusion, our results suggest that HCCR-1 might play a role in the breast tumourigenesis while the overexpression of HCCR-1 induces the obesity probably by inhibiting the cholesterol-lowering effect of ApoE. Therefore, HCCR-1 seems to provide the molecular link between the obesity and the breast cancer risk.

The human cervical cancer oncogene HCCR-1 is overexpressed in various human cancers, and might function as a negative regulator of the p53 tumor suppressor. To determine the regulatory pathway involved in the HCCR-1 gene expression, we searched the 5' flanking region of HCCR-1 and identified HCCR-1 promoter including putative homeodomain protein binding sites. The level of HCCR-1 expression was increased during the mouse embryogenesis. Expression of phosphatidylinositol 3-kinase (PI3K) in NIH/3T3 cells activated the HCCR-1 promoter. This promoter was also activated by wild type Akt but not by dominant negative Akt in K562 cells. In addition, the level of HCCR-1 was decreased by PI3K inhibitor, LY-294002, in a dose dependent manner. Northern blot analysis revealed that the HCCR-1 gene expression was down-regulated by LY-294002. These results suggest that the HCCR-1 oncogene expression was regulated by the PI3K/Akt signaling pathway.

Purpose: The purpose of our study was to identify an unique gene that shows cancer-associated expression, evaluates its potential usefulness in cancer diagnosis, and characterizes its function related to human carcinogenesis. Experimental Design: We used the differential display reverse transcription-PCR method with normal cervical, cervical cancer and metastatic tissues, and cervical cancer cell line to identify genes overexpressed in cancers. Results: We identified a minichromosome maintenance 3 (MCM3) gene that was overexpressed in various human cancers, including leukemia, lymphoma, and carcinomas of the uterine cervix, colon, lung, stomach, kidney and breast, and malignant melanoma. Western blot and immunohistochemical analyses also revealed that MCM3 protein was elevated in most of human cancer tissues tested. We compared the MCM3 protein expression levels in human cancers with conventional proliferation markers, Ki-67 and proliferating cell nuclear antigen. MCM3 antibody was the most specific for multiple human cancers, whereas proliferating cell nuclear antigen was relatively less effective in specificity, and Ki-67 failed to detect several human cancers. The down-regulation of MCM3 protein level was examined under serum starvation in both normal and cancer cells. Interestingly, MCM3 protein was stable in MCF-7 breast cancer cells even up to 96 hours after serum starvation, whereas it was gradually degraded in normal BJ fibroblast cells. Nude mice who received injections of HEK 293 cells stably transfected with MCM3 formed tumors in 6 weeks. Conclusions: Our study indicates that determination of MCM3 expression level will facilitate the assessment of many different human malignancies in tumor diagnosis, and MCM3 is involved in multiple types of human carcino-genesis.

Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of this study was to identify unique oncogenes that are differentially expressed in human cancers and characterize their functions in tumorigenesis. To discover new putative oncogenes, the differential display RT-PCR method was applied using normal cervical tissues, cervical cancer cell lines, cervical cancer tissues, and metastatic tissues. We identified a new human cervical cancer oncogene HCCR-2 that was overexpressed in various human tumors including leukemia, lymphoma, and carcinomas of the breast, kidney, ovary, stomach, colon, and uterine cervix. Ectopic expression of HCCR-2 resulted in direct tumorigenic conversions of NIH/3T3 and Rat1 fibroblasts. Nude mice injected with NIH/3T3 cells stably transfected with HCCR-2 formed tumors in 4 weeks. The resultant tumors display characteristics of an epithelial carcinoma. In HCCR-2 transfected NCI-H460 cells and RKO cells, stabilization of the p53 tumor suppressor occurred without genetic mutation and correlated with functional impairment, as indicated by the defective induction of p53-induced p21(WAF1), MDM2, and bax. These results indicate that HCCR-2 probably represents a new oncogene that is related to tumorigenesis, functioning as a negative regulator of the p53 tumor suppressor.

To identify the genes involved in cervical carcinogenesis, we applied the mRNA differential display method and identified a candidate tumor suppressor gene, HCCS-1, which was present in normal cervical tissue but absent in cervical cancer, metastatic lymph node and CUMC-6 cervical cancer cell line. HCCS-1 transcripts were expressed in many normal tissues including leukocyte, lung, spleen, liver, heart and uterine cervix. Its expression was absent in 8 human cancer cell lines. HCCS-1-transfected HeLa cells exhibited growth inhibition by about 50%. This inhibitory effect of HCCS-1 on cervical cancer cells was associated with apoptotic process including DNA fragmentation. HCCS-1-transfected HeLa cells were shown to release cytochrome c from mitochondria, which activates caspase-9 and -3 and finally results in cleavage of poly(ADP-ribose) polymerase. Apoptosis formation was detected by propidium-iodide/annexin V. HCCS-1-transfected HeLa cells were more sensitive to adriamycin or UVC ray triggered apoptosis. These results suggest that HCCS-1 is downregulated in multiple human tumor types and may serve as a candidate tumor suppressor gene through apoptotic pathway against human cervical cancer. © 2001 Wiley-Liss, Inc.

Background Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules. Results We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell. Conclusions Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

The proliferative capacity of tumor cells is a characteristic feature in the whole growing tumors. Many pathologists and clinicians have used the estimation of cell proliferation for prognostic information. Minichromosome maintenance protein 3 (MCM3) is known to have a role on the initiation and regulation of DNA replication during cell cycle. The aim of this study was to evaluate the potential applicability of one of the MCM proteins, MCM3, as a proliferation marker in papillary thyroid carcinoma (PTC) with correlation to clinicopathological parameters. We performed the immunohistochemical analysis for MCM3 and Ki-67 in 60 cases of PTC and Western blot analysis for MCM3 expression in 6 PTCs and normal thyroid tissues. The comparison of MCM3 labeling index (LI) to tumor size ( P = 0.031) and extrathyroidal extension ( P = 0.037) was statistically significant while that of Ki-67 LI to them was not. Moreover, a significant association was not observed between MCM3 and Ki-67, but the MCM3 LI was considerably higher. Western blot analyses revealed that the MCM3 protein expression levels were overexpressed in all PTCs. On the contrary, the levels of MCM3 were very low or absent in all normal thyroid tissues. Our results indicate that MCM3 may be a more reliable proliferation marker than Ki-67 in accessing the growth of tumor and evaluating tumor aggressiveness of PTC.

Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. HCCR transgenic mice developed breast cancers but it is unknown how HCCR-1 contributes to human tumorigenesis. This study identified a HCCR-1-binding protein 1 (HCCRBP-1) as an HCCR binding partner by performing yeast two hybrid screening. Their endogenous interaction was further confirmed by coimmunoprecipitation experiments. These two proteins colocalized in the mitochondria. HCCRBP-1 was overexpressed in various human tumors. In addition, HCCRBP-1 alone converted NIH/3T3 cells into tumor cells in combination with no other oncogenes. HCCRBP-1 induced tumorigenesis by markedly activating PKC activities but decreasing the pro-apoptotic PKC alpha and PKC delta isoform levels. We observed that p53 stabilization also occurred with functional impairment in HCCRBP-1-transfected 293 cells, as indicated by defective induction of p21, MDM2 and bax. Indeed, HCCRBP-1 decreased p21 promoter activity probably via p53 stabilization leading to the defective function. These results indicate that HCCRBP-1 oncogene induces p53 stabilization and thereby contributes to tumorigenesis.

Background The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene. Results To investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively. Conclusion Our experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation.

Background Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of our study was to identify an unique gene that shows cancer-associated expression, and characterizes its function related to human carcinogenesis. Methods We used the differential display (DD) RT-PCR method using normal cervical, cervical cancer, metastatic cervical tissues, and cervical cancer cell lines to identify genes overexpressed in cervical cancers and identified gremlin 1 which was overexpressed in cervical cancers. We determined expression levels of gremlin 1 using Northern blot analysis and immunohistochemical study in various types of human normal and cancer tissues. To understand the tumorigenesis pathway of identified gremlin 1 protein, we performed a yeast two-hybrid screen, GST pull down assay, and immunoprecipitation to identify gremlin 1 interacting proteins. Results DDRT-PCR analysis revealed that gremlin 1 was overexpressed in uterine cervical cancer. We also identified a human gremlin 1 that was overexpressed in various human tumors including carcinomas of the lung, ovary, kidney, breast, colon, pancreas, and sarcoma. PIG-2-transfected HEK 293 cells exhibited growth stimulation and increased telomerase activity. Gremlin 1 interacted with homo sapiens tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (14-3-3 eta; YWHAH). YWHAH protein binding site for gremlin 1 was located between residues 61–80 and gremlin 1 binding site for YWHAH was found to be located between residues 1 to 67. Conclusion Gremlin 1 may play an oncogenic role especially in carcinomas of the uterine cervix, lung, ovary, kidney, breast, colon, pancreas, and sarcoma. Over-expressed gremlin 1 functions by interaction with YWHAH. Therefore, Gremlin 1 and its binding protein YWHAH could be good targets for developing diagnostic and therapeutic strategies against human cancers.

Purpose: HCCR oncoprotein is reported to be related to tumorigenesis, including breast cancer, functioning as a negative regulator of p53. Mice transgenic for HCCR developed breast cancers. The objective of this study was to validate the HCCR oncoprotein as a candidate biomarker for breast cancer. Experimental Design: HCCR expression in breast cancer cells was analyzed by quantitative PCR, ELISA, immunohistochemistry, Western blotting, fluorescence-activated cell sorting, and confocal microscopy. Epitope areas were determined using mass spectrometry through the analysis of time-dependent tryptic fragment patterns of HCCR. HCCR expression profiles in breast cancer patient sera were analyzed, and correlations with clinicopathologic data and carbohydrate antigen 15-3 (CA15-3) levels were determined. Results: HCCR was up-regulated in breast cancer cells and tissues. The epitope regions of HCCR recognized by monoclonal antibody (BCS-1) were HFWTPK and QQTDFLDIYHAFR. According to fluorescence-activated cell sorting and confocal microscopic analysis, BCS-1 was bound to HCCR antigen on the cell surface. Serum HCCR concentrations were measured using ELISA from 299 subjects, including 129 patients with breast cancer, 24 patients with benign breast disease, and 158 normal volunteers, and comparisons were made to CA15-3. Serologic studies revealed an 86.8% sensitivity for HCCR in breast cancer, which was higher than 21.0% for CA15-3. Eighty-six of 98 (87.8%) patients with breast cancers that were negative for CA15-3 were positive for HCCR-1. A positive response rate of 83.3% was identified even at early stages for pathologic factors in breast cancer. Conclusions: The HCCR assay has an advantage over CA15-3 in diagnosing breast cancer and detecting early stages of the disease.

Transgenic mice containing novel oncogene HCCR-2 were generated to analyse the phenotype and to characterize the role of HCCR-2 in cellular events. Mice transgenic for HCCR-2 developed breast cancers and metastasis. The level of p53 in HCCR-2 transgenic mice was elevated in most tissues including breast, brain, heart, lung, liver, stomach, kidney, spleen, and lymph node. We examined whether stabilized p53 is functional in HCCR-2 transgenic mice. Defective induction of p53 responsive genes including p21WAF1, MDM2, and bax indicates that stabilized p53 in HCCR-2 transgenic mice exists in an inactive form. These results suggest that HCCR-2 represents an oncoprotein that is related to breast cancer development and regulation of the p53 tumor suppressor.