Significance of combination therapy of zoledronic acid and gemcitabine on pancreatic cancer.

In the present study, we examined the cytotoxic effects of combination therapy with zoledronic acid (ZOL) and gemcitabine (GEM) on pancreatic cancer cells in vitro and in vivo. Four human pancreatic cancer cell lines were treated with ZOL, GEM or a combination of both, and the effects of the respective drug regimens on cell proliferation, invasion and matrix metalloproteinase (MMP) expression were examined. A pancreatic cancer cell line was also intrasplenically or orthotopically implanted into athymic mice and the effects of these drugs on tumor metastasis and growth in vivo were evaluated by histological and immunohistochemical analyses. Combination treatment with low doses of ZOL and GEM efficiently inhibited the proliferation (P < 0.001) and invasion (P < 0.001) of pancreatic cancer cells in vitro. Western blotting assay revealed that MMP-2 and MMP-9 expression levels were decreased after ZOL treatment. In vivo, combined treatment significantly inhibited tumor growth (P < 0.05) and the development of liver metastasis (P < 0.05). These data revealed that ZOL and GEM, when used in combination, have significant antitumor, anti-metastatic and anti-angiogenic effects on pancreatic cancer cells. The present study is the first to report the significance of the combination treatment of ZOL and GEM in pancreatic cancer using an in vivo model. These data are promising for the future application of this drug regimen in patients with pancreatic cancer.

Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer­associated fibroblasts.

Cancer­associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor­stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.

MUTYH-null mice are susceptible to spontaneous and oxidative stress induced intestinal tumorigenesis.

MUTYH is a mammalian DNA glycosylase that initiates base excision repair by excising adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine, thereby preventing G:C to T:A transversion caused by oxidative stress. Recently, biallelic germ-line mutations of MUTYH have been found in patients predisposed to a recessive form of hereditary multiple colorectal adenoma and carcinoma with an increased incidence of G:C to T:A somatic mutations in the APC gene. In the present study, a systematic histologic examination revealed that more spontaneous tumors had developed in MUTYH-null mice (72 of 121; 59.5%) than in the wild type (38 of 109; 34.9%). The increased incidence of intestinal tumors in MUTYH-null mice (11 tumors in 10 of 121 mice) was statistically significant compared with the wild type (no intestinal tumors in 109 mice). Two adenomas and seven adenocarcinomas were observed in the small intestines, and two adenomas but no carcinomas were found in the colons. In MUTYH-null mice treated with KBrO(3), the occurrence of small intestinal tumors dramatically increased. The mean number of polyps induced in the small intestines of these mice was 61.88 (males, 72.75; females, 51.00), whereas it was 0.85 (males, 0.50; females, 1.00) in wild-type mice. The tumors developed predominantly in the duodenum and in the upper region of the (jejunum) small intestines. We conclude that MUTYH suppresses spontaneous tumorigenesis in mammals, thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and a recessive form of human hereditary colorectal adenoma and carcinoma.

Mucoepidermoid carcinoma of the anal canal: A case report and review of the literature.

Mucoepidermoid carcinoma of the anal canal is a rare tumor. We herein report the case of a 74-year-old male patient with a high-grade mucoepidermoid carcinoma of the anal canal who was treated by local surgical resection and subsequent irradiation. However, the patient succumbed to liver and lung metastases 2 years after the procedure. The characteristic findings of mucoepidermoid carcinoma of the anus remain unclear to date due to rarity of this tumor. Since 1954, when this type of tumor was first described, only 58 cases of patients diagnosed with mucoepidermoid carcinoma of the anus have been reported to date. In this context, a review of the existing English literature on this rare tumor was also performed.

Anal Canal Duplication Associated with Presacral Cyst in an Adult.

Anal canal duplication (ACD) is a rare congenital malformation, usually detected early in life. We report a case of a 67-year-old female with symptomatic ACD associated with a presacral cyst. Physical examination revealed an accessory opening located in the midline, posterior to the true anus. Imaging examinations, including fistulography, endoanal ultrasonography, and magnetic resonance imaging, revealed a blind-ending fistulous tract without connecting with the rectum and a presacral cyst posterior to the rectum. Complete surgical excision of the tract with cyst was performed through a posterior sagittal approach. Histologic examination revealed squamous epithelium lining and smooth muscle bundles, thereby confirming ACD. The postoperative course was uneventful, and the patient was doing well; no recurrence was observed 4 years after surgery. ACD can present for the first time in infants, children, and adults. Imaging examinations are useful for the diagnosis and preoperative assessment of ACD. Therefore, ACD should be considered in the differential diagnosis, even in adults, when a posterior perineal orifice is encountered, particularly in female patients. Once ACD is suspected, intense imaging inspection is recommended to visualize the ACD and associated anomalies, and surgical removal is warranted to prevent inflammatory complications or malignant changes.

Replication-associated repair of adenine:8-oxoguanine mispairs by MYH.

Cellular DNA is constantly exposed to the risk of oxidation. 8-oxoguanine (8-oxoG) is one of the major DNA lesions generated by oxidation, which is primarily corrected by base excision repair. When it is not repaired prior to replication, replicative DNA polymerases yield misinsertion of an adenine (A) opposite the 8-oxoG on the template strand, generating an A:8-oxoG mispair. MYH, a mammalian homolog of Escherichia coli MutY, is a DNA glycosylase responsible for initiating base excision repair of such a mispair by excising the adenine opposite 8-oxoG. Here, using an in vivo repair system, we show that DNA replication enhances the repair of the A:8-oxoG mispair. Repair efficiency was lower in MYH-deficient murine cells than in MYH-proficient cells. Transfection of the MYH-deficient cells with a wild-type MYH expression vector increased the efficiency of A:8-oxoG repair, indicating that a significant part of this replication-associated repair depends on MYH. Expression of a mutant MYH in which the PCNA binding motif was disrupted did not increase the repair efficiency, thus suggesting that the interaction between PCNA and MYH is critical for MYH-initiated repair of A:8-oxoG.

A functional analysis of the DNA glycosylase activity of mouse MUTYH protein excising 2-hydroxyadenine opposite guanine in DNA.

2-hydroxy-2-deoxyadenosine triphosphate (2-OH-dATP), generated by the oxidation of dATP, can be misincorporated by DNA polymerases opposite guanine in template DNA during DNA replication, thus causing spontaneous mutagenesis. We demonstrated that mouse MUTYH (mMUTYH) has a DNA glycosylase activity excising not only adenine opposite 8-oxoguanine (8-oxoG) but also 2-hydroxyadenine (2-OH-A) opposite guanine, using purified recombinant thioredoxin-mMUTYH fusion protein. mMUTYH formed a stable complex with duplex oligonucleotides containing an adenine:8-oxoG pair, but the binding of mMUTYH to oligonucleotides containing a 2-OH-A:guanine pair was barely detectable, thus suggesting that mMUTYH recognizes and interacts with these two substrates in a different manner which may reflect the difference in the base excision repair process for each substrate. Mutant mMUTYH with G365D amino acid substitution, corresponding to a G382D germline mutation of human MUTYH found in familial adenomatous polyposis patients, almost completely retained its DNA glycosylase activity excising adenine opposite 8-oxoG; however, it possessed 1.5% of the wild-type activity excising 2-OH-A opposite guanine. Our results imply that the reduced repair capacity of the mutant hMUTYH(G382D), which inefficiently excises 2-OH-A opposite guanine, results in an increased occurrence of somatic G:C to T:A transversion mutations in the APC gene as well as tumorigenesis in the colon.

8-Oxoguanine formation induced by chronic UVB exposure makes Ogg1 knockout mice susceptible to skin carcinogenesis.

8-Oxoguanine is one of the oxidative DNA damages that can result in stable mutations. The Ogg1 gene encodes the repair enzyme 8-oxoguanine-DNA glycosylase, which removes the oxidized base from DNA. In this study, we investigated the role of 8-oxoguanine in skin carcinogenesis induced by UVB irradiation using Ogg1 knockout mice (C57Bl/6J background). We examined the effect of UVB irradiation on the formation of 8-oxoguanine in epidermal cells using immunostaining and found that the level of 8-oxoguanine in Ogg1 knockout mice 24 hours after UVB irradiation remained high compared with that in wild-type and heterozygous mice. To verify the effect of chronic UVB irradiation on 8-oxoguanine formations in epidermal cells, we irradiated wild-type, heterozygous, and Ogg1 knockout mice with UVB at a dose of 2.5 kJ/m2 thrice a week for 40 weeks. We found that the mean number of tumors in Ogg1 knockout mice was 3.71, which was significantly more than in wild-type and heterozygous mice, being 1.71 and 2.28, respectively. The rate of developing malignant tumors in Ogg1 knockout mice was also significantly higher (88.5%; squamous cell carcinomas, 73.1%; sarcomas, 15.4%) than in wild-type mice (50.0%; squamous cell carcinomas, 41.7%; sarcomas, 8.3%). Moreover, the age of onset of developing skin tumors in Ogg1 knockout mice was earlier than in the other types of mice. These results clearly indicate that oxidative DNA damage induced by sunlight plays an important role in the development of skin cancers.

Endoanal Ultrasonography of Mucinous Adenocarcinoma Arising from Chronic Fistula-in-ano: Three Case Reports.

Mucinous adenocarcinoma arising in chronic fistula-in-ano is rare, and diagnosing it at an early stage is difficult. The role of endoanal ultrasonography in diagnosing the condition has not been discussed in the study. Herein, we report three cases of mucinous adenocarcinoma arising from anal fistulas in which endosonography played an important role in diagnosing malignant change. Three male patients with a 5- to 20-year history of anal fistula were referred to our hospital due to perianal induration, progressive anal pain, or mucopurulent secretion. In all three patients, endosonography revealed a multiloculated complex echoic mass with isoechoic solid components communicating with a trans-sphincteric fistula and sonography-guided biopsy under anesthesia revealed mucinous adenocarcinoma. All patients underwent abdominoperineal resection with lymph node dissection. One patient with a local recurrence died 3 years after surgery and two have remained disease-free for >6 years. These observations suggest that endosonography may be a reliable technique for the diagnosis of mucinous adenocarcinoma arising from chronic fistula-in-ano. Sonography-guided biopsy is useful for the definitive diagnosis of malignancy. Therefore, periodic endosonography assessment should be recommended for patients with persistent anal fistula, especially those with progressive clinical symptoms. Once malignancy is suspected, aggressive sonography-guided biopsy under anesthesia should be performed, which may enable an early diagnosis, curative treatment, and favorable long-term results.

Degree of desmoplasia in metastatic lymph node lesions is associated with lesion size and poor prognosis in pancreatic cancer patients.

Pancreatic cancer is characterized by increased hyperplasia of fibrotic tissue, termed desmoplasia, and lymph node metastasis is an independent prognostic factor in this disease. However, there are no reports focused on desmoplasia in pancreatic cancer lymph node metastases. The present study evaluated a range of factors and investigated their association with poor prognosis in pancreatic cancer cases with lymph node metastasis, including the degree of desmoplasia in lesions. To identify the poor prognostic factors associated with lymph node metastasis, the present study retrospectively reviewed the clinical data of 65 patients with lymph node metastases that underwent surgical pancreatic cancer resection between 2007 and 2012 at a single institution. The investigation focused on the degree of fibrosis in metastatic lesions in 216 lymph nodes, and investigated associations with prognosis or clinicopathological findings. The ratios of the fibrotic area in metastatic lymph node lesions were evaluated and classified into three categories, high (≥70%), moderate (10-70%) and low (<10%). Desmoplasia was not observed in cancer-free lymph nodes. The size of metastatic lymph node lesions was additionally measured, and a significant association between metastatic lesion size and the degree of desmoplasia was observed (P<0.001). The degree of desmoplasia was additionally associated with local extranodal invasion. In the analysis of 65 pancreatic cancer patients with metastatic lymph nodes, the presence of multiple metastatic lymph nodes with moderate or high desmoplasia was significantly associated with poor survival (high, P=0.0048; moderate/high, P=0.0075). Of several clinicopathological factors, the presence of multiple metastatic lymph nodes with high or moderate desmoplasia was associated with overall survival in univariate (P=0.0098) and multivariate (P=0.0466) analyses. The degree of desmoplasia in metastatic lymph nodes is associated with lesion size, and the presence of multiple metastatic lymph nodes with desmoplasia is an independent poor prognostic factor, suggesting that the desmoplasia may have an important role in the malignant progression of lymph node metastases.

Futile short-patch DNA base excision repair of adenine:8-oxoguanine mispair.

8-oxo-7, 8-dihydrodeoxyguanosine (8-oxo-dG), one of the representative oxidative DNA lesions, frequently mispairs with the incoming dAMP during mammalian DNA replication. Mispaired dA is removed by post-replicative base excision repair (BER) initiated by adenine DNA glycosylase, MYH, creating an apurinic (AP) site. The subsequent mechanism ensuring a dC:8-oxo-dG pair, a substrate for 8-oxoguanine DNA glycosylase (OGG1), remains to be elucidated. At the nucleotide insertion step, none of the mammalian DNA polymerases examined exclusively inserted dC opposite 8-oxo-dG that was located in a gap. AP endonuclease 1, which possesses 3'-->5' exonuclease activity and potentially serves as a proofreader, did not discriminate dA from dC that was located opposite 8-oxo-dG. However, human DNA ligases I and III joined 3'-dA terminus much more efficiently than 3'-dC terminus when paired to 8-oxo-dG. In reconstituted short-patch BER, repair products contained only dA opposite 8-oxo-dG. These results indicate that human DNA ligases discriminate dC from dA and that MYH-initiated short-patch BER is futile and hence this BER must proceed to long-patch repair, even if it is initiated as short-patch repair, through strand displacement synthesis from the ligation-resistant dC terminus to generate the OGG1 substrate, dC:8-oxo-dG pair.

MUTYH prevents OGG1 or APEX1 from inappropriately processing its substrate or reaction product with its C-terminal domain.

MutY homolog (MUTYH) excises adenine opposite 8-oxoguanine (8-oxoG) in DNA, thus preventing occurrence of G:C to T:A transversion. In cell-free extract prepared from the thymocytes of wild type but not MUTYH-null mice, adenine opposite 8-oxoG in DNA was excised by MUTYH, however, the generated apurinic (AP) site opposite 8-oxoG mostly remained unincised. Recombinant mouse MUTYH (mMUTYH) efficiently excised adenine opposite 8-oxoG and prevented mouse AP endonuclease (mAPEX1) from incising the generated AP site. In contrast, an AP site opposite 8-oxoG created by uracil DNA glycosylase or tetrahydrofuran opposite 8-oxoG was efficiently incised by mAPEX1 in the presence of an excess amount of mMUTYH. Mutant mMUTYH with R361A or G365D substitution, excised adenine opposite 8-oxoG as efficiently as did wild-type mMUTYH, but failed to prevent mAPEX1 from incising the generated AP site. Wild-type mMUTYH bound duplex oligonucleotides containing A:8-oxoG pair with a lower apparent K(d) than that of the mutants, and prevented OGG1 from excising 8-oxoG opposite adenine or the generated AP site. The G365D mutant failed to prevent OGG1 from excising 8-oxoG opposite the generated AP site, thus indicating that the protection of its own product by mMUTYH is an intrinsic function which depends on the C-terminal domain of mMUTYH.

Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts.

There are three types of mouse Mutyh mRNAs (type a, b and c) generated by alternative splicing, and type b mRNA is a major form among the three in most of the tissues examined. The level of type c mRNA is relatively high in brain. Type a and b mRNAs were expected to encode 57.7 kDa protein (MUTYHalpha), while type c mRNA had a partly different open reading frame encoding a 50.2 kDa protein (MUTYHbeta). An in vitro translation of type b and c mRNAs produced a 50 kDa MUTYHalpha and 47 kDa MUTYHbeta, respectively. MUTYHalpha and MUTYHbeta were detected in wild-type embryonic stem (ES) cells or thymocytes prepared from wild-type mice, but neither MUTYH-null ES cells nor thymocytes prepared from MUTYH-null mice. Both MUTYHalpha and MUTYHbeta were mainly localized in the nuclei and some in mitochondria in wild-type ES cells. Recombinant MUTYHalpha and beta were expressed as fusion proteins with thioredoxin in Escherichia coli, but only MUTYHalpha was partly soluble and thus could be purified. Recombinant MUTYHalpha possessed DNA glycosylase activities to excise adenine opposite 8-oxoguanine and guanine but not AP lyase activity.

Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption.

Using Mth1 and Ogg1 knockout mice, we evaluated the roles of these enzymes to prevent tumorigenesis and the accumulation of 8-oxoguanine (8-oxoG) in DNA. We found that lung adenoma/carcinoma spontaneously developed in Ogg1 knockout mice approximately 1.5 years after birth in which 8-oxoG was found to accumulate in their genomes. The mean number of tumors/mouse was 0.71 for the Ogg1 knockout mice, which was five times higher than that observed in wild-type mice (0.14). Although the accumulation of 8-oxoG was also confirmed in the Ogg1, Mth1 double knockout mice, we found no tumor in the lungs of these mice. This observation suggests that Mth1 gene disruption resulted in a suppression of the tumorigenesis caused by an Ogg1 deficiency.

Accumulation of 8-oxoguanine in the cellular DNA and the alteration of the OGG1 expression during ischemia-reperfusion injury in the rat kidney.

During ischemia-reperfusion (I/R) injury in the rat kidney, apoptosis was observed in the distal tubules of the cortico-medullary region and outer medulla (OM) while severe necrosis was seen in the proximal straight tubules of the OM. The majority of these changes disappeared within 2 weeks. We examined the contents of 8-oxo-2'-deoxyguanosine (8-oxo-dG), which is a major type of oxidative damage in DNA, in the rat kidney during I/R injury, and also investigated the expression level of the OGG1 gene encoding the 8-oxoguanine DNA glycosylase. High-performance liquid chromatography with an MS/MS analysis of the nuclear DNA revealed an immediate accumulation of 8-oxo-dG in the nuclear DNA prepared from the cortex and OM of the kidney 1h after I/R, and an immunohistochemical analysis demonstrated the immediate accumulation of 8-oxo-dG in the nuclei of renal tubular cells both in the cortex and OM. A delayed increase of cytoplasmic staining with anti-8-oxo-dG was observed only in the cortico-medulla and OM, where the cytoplasmic staining in the proximal tubular cells is higher than in the distal tubular cells. The level of cytoplasmic staining representing 8-oxo-dG in mitochondrial DNA, peaked at 6h after I/R and preceded the necrosis of proximal tubular cells in the OM. An RNase protection assay showed a high level of OGG1 mRNA in the normal kidney, and the level decreased within 3h only in the OM, and increased thereafter 1-7 days of I/R both in the cortex and OM. In situ hybridization showed higher levels of OGG1 mRNA expression in the renal tubules in the OM than in the cortex of the normal kidney, which decreased rapidly within 3h of I/R. Thus, the accumulation of 8-oxo-dG in the mitochondrial DNA rather than in nuclear DNA is likely to be involved in the pathogenic responses such as necrosis of renal tubular cells during I/R injury of the kidney, together with an altered level of OGG1 expression.

Biological significance of the defense mechanisms against oxidative damage in nucleic acids caused by reactive oxygen species: from mitochondria to nuclei.

In mammalian cells, more than one genome in a single cell has to be maintained throughout the entire life of the cell, namely, one in the nucleus and the other in the mitochondria. The genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are inevitably generated as a result of the respiratory function in mitochondria. To counteract such oxidative damage in nucleic acids, cells are equipped with several defense mechanisms. Modified nucleotides in the nucleotide pools are hydrolyzed, thus avoiding their incorporation into DNA or RNA. Damaged bases in DNA with relatively small chemical alterations are mainly repaired by the base excision repair (BER) system, which is initiated by the excision of damaged bases by specific DNA glycosylases. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP, 8-oxo-dATP, and 2-hydroxy (OH)-dATP to the monophosphates, and MTH1 are located in the cytoplasm, mitochondria, and nucleus. We observed an increased susceptibility to spontaneous carcinogenesis in Mth1-deficient mice and an alteration of MTH1 expression along with the accumulation of 8-oxo-dG in patients with various neurodegenerative diseases. Enzymes for the BER pathway, namely, 8-oxoG DNA glycosylase (OGG1), 2-OH-A/adenine DNA glycosylase (MUTYH), and AP endonuclease (APEX2) are also located both in the mitochondria and in the nuclei, and the expression of mitochondrial OGG1 is altered in patients with various neurodegenerative diseases. We also observed increased susceptibilities to spontaneous carcinogenesis in OGG1 and MUTYH-deficient mice. The increased occurrence of lung tumor in OGG1-deficient mice was completely abolished by the concomitant disruption of the Mth1 gene.

FosB is essential for the enhancement of stress tolerance and antagonizes locomotor sensitization by ΔFosB.

BACKGROUND: Molecular mechanisms underlying stress tolerance and vulnerability are incompletely understood. The fosB gene is an attractive candidate for regulating stress responses, because ΔFosB, an alternative splice product of the fosB gene, accumulates after repeated stress or antidepressant treatments. On the other hand, FosB, the other alternative splice product of the fosB gene, expresses more transiently than ΔFosB but exerts higher transcriptional activity. However, the functional differences of these two fosB products remain unclear. METHODS: We established various mouse lines carrying three different types of fosB allele, wild-type (fosB(+)), fosB-null (fosB(G)), and fosB(d) allele, which encodes ΔFosB but not FosB, and analyzed them in stress-related behavioral tests. RESULTS: Because fosB(+/d) mice show enhanced ΔFosB levels in the presence of FosB and fosB(d/d) mice show more enhanced ΔFosB levels in the absence of FosB, the function of FosB can be inferred from differences observed between these lines. The fosB(+/d) and fosB(d/d) mice showed increased locomotor activity and elevated Akt phosphorylation, whereas only fosB(+/d) mice showed antidepressive-like behaviors and increased E-cadherin expression in striatum compared with wild-type mice. In contrast, fosB-null mice showed increased depression-like behavior and lower E-cadherin expression. CONCLUSIONS: These findings indicate that FosB is essential for stress tolerance mediated by ΔFosB. These data suggest that fosB gene products have a potential to regulate mood disorder-related behaviors.

Prospectively isolated cancer-associated CD10(+) fibroblasts have stronger interactions with CD133(+) colon cancer cells than with CD133(-) cancer cells.

Although CD133 has been reported to be a promising colon cancer stem cell marker, the biological functions of CD133+ colon cancer cells remain controversial. In the present study, we investigated the biological differences between CD133+ and CD133- colon cancer cells, with a particular focus on their interactions with cancer-associated fibroblasts, especially CD10+ fibroblasts. We used 19 primary colon cancer tissues, 30 primary cultures of fibroblasts derived from colon cancer tissues and 6 colon cancer cell lines. We isolated CD133+ and CD133- subpopulations from the colon cancer tissues and cultured cells. In vitro analyses revealed that the two populations showed similar biological behaviors in their proliferation and chemosensitivity. In vivo analyses revealed that CD133+ cells showed significantly greater tumor growth than CD133- cells (P=0.007). Moreover, in cocultures with primary fibroblasts derived from colon cancer tissues, CD133+ cells exhibited significantly more invasive behaviors than CD133- cells (P<0.001), especially in cocultures with CD10+ fibroblasts (P<0.0001). Further in vivo analyses revealed that CD10+ fibroblasts enhanced the tumor growth of CD133+ cells significantly more than CD10- fibroblasts (P<0.05). These data demonstrate that the in vitro invasive properties and in vivo tumor growth of CD133+ colon cancer cells are enhanced in the presence of specific cancer-associated fibroblasts, CD10+ fibroblasts, suggesting that the interactions between these specific cell populations have important roles in cancer progression. Therefore, these specific interactions may be promising targets for new colon cancer therapies.

Antagonistic regulation of cell-matrix adhesion by FosB and DeltaFosB/Delta2DeltaFosB encoded by alternatively spliced forms of fosB transcripts.

Among fos family genes encoding components of activator protein-1 complex, only the fosB gene produces two forms of mature transcripts, namely fosB and DeltafosB mRNAs, by alternative splicing of an exonic intron. The former encodes full-length FosB. The latter encodes DeltaFosB and Delta2DeltaFosB by alternative translation initiation, and both of these lack the C-terminal transactivation domain of FosB. We established two mutant mouse embryonic stem (ES) cell lines carrying homozygous fosB-null alleles and fosB(d) alleles, the latter exclusively encoding DeltaFosB/Delta2DeltaFosB. Comparison of their gene expression profiles with that of the wild type revealed that more than 200 genes were up-regulated, whereas 19 genes were down-regulated in a DeltaFosB/Delta2DeltaFosB-dependent manner. We furthermore found that mRNAs for basement membrane proteins were significantly up-regulated in fosB(d/d) but not fosB-null mutant cells, whereas genes involved in the TGF-beta1 signaling pathway were up-regulated in both mutants. Cell-matrix adhesion was remarkably augmented in fosB(d/d) ES cells and to some extent in fosB-null cells. By analyzing ES cell lines carrying homozygous fosB(FN) alleles, which exclusively encode FosB, we confirmed that FosB negatively regulates cell-matrix adhesion and the TGF-beta1 signaling pathway. We thus concluded that FosB and DeltaFosB/Delta2DeltaFosB use this pathway to antagonistically regulate cell matrix adhesion.