Osteopontin (OPN) is a phosphorylated glycoprotein that binds to a v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

Effective inhibitors of osteopontin (OPN) -mediated neoplastic transformation and metastasis are still lacking. (-)-Agelastatin A is a naturally occurring oroidin alkaloid with powerful antitumor effects that, in many cases, are superior to cisplatin in vitro . In this regard, past comparative assaying of the two agents against a range of human tumor cell lines has revealed that typically (-)-agelastatin A is 1.5 to 16 times more potent than cisplatin at inhibiting cell growth, its effects being most pronounced against human bladder, skin, colon, and breast carcinomas. In this study, we have investigated the effects of (-)-agelastatin A on OPN-mediated malignant transformation using mammary epithelial cell lines. Treatment with (-)-agelastatin A inhibited OPN protein expression and enhanced expression of the cellular OPN inhibitor, Tcf-4. (-)-Agelastatin A treatment also reduced B-catenin protein expression and reduced anchorage-independent growth, adhesion, and invasion in R37 OPN pBK-CMV and C9 cell lines. Similar effects were observed in MDA-MB-231 and MDA-MB-435s human breast cancer cell lines exposed to (-)-agelastatin A. Suppression of Tcf-4 by RNA interference (short interfering RNA) induced malignant/invasive transformation in parental benign Rama 37 cells; significantly, these events were reversed by treatment with (-)-agelastatin A. Our study reveals, for the very first time, that (-)-agelastatin A down-regulates B-catenin expression while simultaneously up-regulating Tcf-4 and that these combined effects cause repression of OPN and inhibition of OPN-mediated malignant cell invasion, adhesion, and colony formation in vitro. We have also shown that (-)-agelastatin A inhibits cancer cell proliferation by causing cells to accumulate in the G 2 phase of cell cycle.

Graphical abstract

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor ␣, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorageindependent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.

The synthesis of three potent new antitumor agents is described: the A83586C-citropeptin hybrid (1), the A83586C-GE3 hybrid (2), and l-Pro-A83586C (3). Significantly, compounds 1 and 2 function as highly potent inhibitors of beta-catenin/TCF4 signaling within cancer cells, while simultaneously downregulating osteopontin (Opn) expression. A83586C antitumor cyclodepsipeptides also inhibit E2F-mediated transcription by downregulating E2F1 expression and inducing dephosphorylation of the oncogenic hyperphosphorylated retinoblastoma protein (pRb).

Red meat consumption is associated with endogenous metabolic generation of mutagenic N-nitroso compounds (NOC) and may be implicated in causation of colorectal cancer. Assessment of a biologically relevant dose of NOCs is hampered by imperfect understanding of NOC interactions with other dietary components. This study tests the hypothesis that NOC effects upon mutational biomarkers in mouse colon may be modulated by a non-genotoxic diet-related compound. N-methyl-N-nitrosourea (MNU) and undegraded lambda carrageenan (lambdaCgN) were selected as test chemicals, representing a NOC and a non-genotoxic agent, respectively. Study end-points included (i) DNA adduct formation and (ii) metallothionein (MT) crypt restricted immunopositivity indices (MTCRII) which are considered representative of crypt stem cell mutations. Frequency and size of MT immunopositive foci as well as total number of MT immunopositive crypts were assessed. Biologically effective doses of MNU and lambdaCgN were determined in model validation studies and the agents were then tested alone and in combination. Continuous lambdaCgN treatment for 10 weeks induced significantly greater colonic mucosal injury than a drinking water control. In combined treatment regimens, lambdaCgN treatment did not significantly affect MNU-induced DNA adduct formation. However, combinations of lambdaCgN with MNU significantly increased MTCRII in excess of those induced by MNU alone. Recurrent or continuous lambdaCgN regimens had greater interactive effects with MNU upon MTCRII than short-term lambdaCgN treatment. This study has shown that exposure to a non-genotoxic diet-related compound (lambdaCgN) modulates the effective NOC dosimetry for induction of MT crypt restricted immunopositivity.

Obese AT (adipose tissue) exhibits increased macrophage number. Pro-inflammatory CD16+ peripheral monocyte numbers are also reported to increase with obesity. The present study was undertaken to simultaneously investigate obesity-associated changes in CD16+ monocytes and ATMs (AT macrophages). In addition, a pilot randomized placebo controlled trial using the PPAR (peroxisome-proliferator-activated receptor) agonists, pioglitazone and fenofibrate was performed to determine their effects on CD14+/CD16+ monocytes, ATM and cardiometabolic and adipose dysfunction indices. Obese glucose-tolerant men (n=28) were randomized to placebo, pioglitazone (30 mg/day) and fenofibrate (160 mg/day) for 12 weeks. A blood sample was taken to assess levels of serum inflammatory markers and circulating CD14+/CD16+ monocyte levels via flow cytometry. A subcutaneous AT biopsy was performed to determine adipocyte cell surface and ATM number, the latter was determined via assessment of CD68 expression by IHC (immunohistochemistry) and real-time PCR. Subcutaneous AT mRNA expression of CEBPβ (CCAAT enhancer-binding protein β), SREBP1c (sterol-regulatory-element-binding protein 1c), PPARγ2, IRS-1 (insulin receptor substrate-1), GLUT4 (glucose transporter type 4) and TNFα (tumour necrosis factor α) were also assessed. Comparisons were made between obese and lean controls (n=16) at baseline, and pre- and post-PPAR agonist treatment. Obese individuals had significantly increased adipocyte cell surface, percentage CD14+/CD16+ monocyte numbers and ATM number (all P=0.0001). Additionally, serum TNF-α levels were significantly elevated (P=0.017) and adiponectin levels reduced (total: P=0.0001; high: P=0.022) with obesity. ATM number and percentage of CD14+/CD16+ monocytes correlated significantly (P=0.05). Pioglitazone improved adiponectin levels significantly (P=0.0001), and resulted in the further significant enlargement of adipocytes (P=0.05), without effect on the percentage CD14+/CD16+ or ATM number. Pioglitazone treatment also significantly increased subcutaneous AT expression of CEBPβ mRNA. The finding that improvements in obesity-associated insulin resistance following pioglitazone were associated with increased adipocyte cell surface and systemic adiponectin levels, supports the centrality of AT to the cardiometabolic derangement underlying the development of T2D (Type 2 diabetes) and CVD (cardiovascular disease).

Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (lCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded lCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by lCgN alone. Combined lCgN/MNU treatments induced greater MTCRII (Po0.01) as well as greater number (Po0.001) and crypt multiplicity (Po0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r ¼ 0.732; Po0.01). MTCRII are induced by lCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel mediumterm murine bioassay relevant to early-stage colorectal tumorigenesis.