PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer.

CD73 is a cell surface ecto-5'-nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5'-(α, ß-methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial-mesenchymal transition.

Rebound increases in chemokines by CXCR2 antagonist in breast cancer can be prevented by PKCδ and PKCε activators.

Activation of CXCR2 by chemokines such as CXCL1 and CXCL2 increases aggressiveness of breast cancer, inducing chemoresistance, hence CXCR2 antagonists are in clinical trials. We previously reported that inhibition of CXCR2 increases MIP-2 (CXCL2), which may inhibit anti-tumoral effects of CXCR2 antagonists. This seems to be due to inhibition of protein kinase C (PKC) by CXCR2 antagonist since specific inhibitor of PKC also enhances MIP-2 secretion. We here examined whether CXCR2 inhibitor also increases KC (CXCL1) secretion, ligand for CXCR2 involved in metastasis and PKC activators can prevent increases in chemokine secretion. We used SB 225002, which is a specific CXCR2 antagonist. The effects of PKC activators that have documented anti-tumoral effects and activates multiple isozymes of PKC such as Ingenol-3-angelate (I3A) and bryostatin-1 were examined here. In addition, FR236924, PKCε selective and 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), PKCδ selective activators were also tested. The effects of activators were determined using brain metastatic (4TBM) and heart metastatic (4THM) subset of 4T1 breast carcinoma cells because these aggressive carcinoma cells with cancer stem cell features secrete high levels of KC and MIP-2. Inhibition of CXCR-2 activity increased KC (CXCL1) secretion. PKC activators prevented SB225002-induced increases in KC and MIP-2 secretion. Different activators/modulators induce differential changes in basal and SB225002-induced chemokine secretion as well as cell proliferation and the activators that act on PKCδ and/or PKCε such as bryostatin 1, FR236924 and Roy-Bz are the most effective. These activators alone also decrease cell proliferation or chemokine secretion or both. Given the role of KC and MIP-2 in drug resistance including chemotherapeutics, activators of PKCε and PKCδ may prevent emerging of resistance to CXCR2 inhibitors as well as other chemotherapeutics.

G9a Promotes Breast Cancer Recurrence through Repression of a Pro-inflammatory Program.

Dysregulated gene expression is a common feature of cancer and may underlie some aspects of tumor progression, including tumor relapse. Here, we show that recurrent mammary tumors exhibit global changes in gene expression and histone modifications and acquire dependence on the G9a histone methyltransferase. Genetic ablation of G9a delays tumor recurrence, and pharmacologic inhibition of G9a slows the growth of recurrent tumors. Mechanistically, G9a activity is required to silence pro-inflammatory cytokines, including tumor necrosis factor (TNF), through H3K9 methylation at gene promoters. G9a inhibition induces re-expression of these cytokines, leading to p53 activation and necroptosis. Recurrent tumors upregulate receptor interacting protein kinase-3 (RIPK3) expression and are dependent upon RIPK3 activity. High RIPK3 expression renders recurrent tumors sensitive to necroptosis following G9a inhibition. These findings demonstrate that G9a-mediated silencing of pro-necroptotic proteins is a critical step in tumor recurrence and suggest that G9a is a targetable dependency in recurrent breast cancer.

Limiting Self-Renewal of the Basal Compartment by PKA Activation Induces Differentiation and Alters the Evolution of Mammary Tumors.

Differentiation therapy utilizes our understanding of the hierarchy of cellular systems to pharmacologically induce a shift toward terminal commitment. While this approach has been a paradigm in treating certain hematological malignancies, efforts to translate this success to solid tumors have met with limited success. Mammary-specific activation of PKA in mouse models leads to aberrant differentiation and diminished self-renewing potential of the basal compartment, which harbors mammary repopulating cells. PKA activation results in tumors that are more benign, exhibiting reduced metastatic propensity, loss of tumor-initiating potential, and increased sensitivity to chemotherapy. Analysis of tumor histopathology revealed features of overt differentiation with papillary characteristics. Longitudinal single-cell profiling at the hyperplasia and tumor stages uncovered an altered path of tumor evolution whereby PKA curtails the emergence of aggressive subpopulations. Acting through the repression of SOX4, PKA activation promotes tumor differentiation and represents a possible adjuvant to chemotherapy for certain breast cancers.

Cell-Autonomous versus Systemic Akt Isoform Deletions Uncovered New Roles for Akt1 and Akt2 in Breast Cancer.

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.

Global gene expression profile in canine mammary carcinomas.

Mammary gland tumors are a heterogeneous group of neoplastic diseases. Genetic studies make it possible to determine genetic profiles and identify new molecular markers. The aim of the study was to evaluate the gene expression profile of canine mammary carcinomas and identify potential prognostic markers. Twelve mammary cancer samples from bitches were collected for the evaluation of global gene expression. Microarray assays were performed using commercial kits. Statistical analysis of the microarray was done using moderate t-statistic and adjusted using the Benjamini and Hochberg procedure. Differential connectivity analysis was also performed. Enrichment analyses were conducted using WebGestalt. P-values were calculated using hypergeometric statistics and adjusted using the Benjamini and Hochberg procedure. The HYAL-1 gene was validated using quantitative PCR (qPCR). There were 878 upregulated genes and 821 downregulated genes in the neoplasms studied. Enrichment analysis (individual analysis) identified the HYAL-1 gene as a potential marker of tumorigenesis and tumor recurrence. Differential connectivity analysis demonstrated 262 differentially connected genes.

Significance of sphingosine kinase 1 expression in feline mammary tumors.

BACKGROUND: Sphingosine kinase 1 (SPHK1) is an enzyme that converts pro-apoptotic ceramide and sphingosine into anti-apoptotic sphingosine-1-phosphate. There is growing evidence that SPHK1 activation promotes oncogenic transformation, tumor growth, chemotherapy resistance, and metastatic spread. High SPHK1 expression has been associated with a poor prognosis in several human cancers. RESULTS: In the present study, the expression level of SPHK1 was examined in feline mammary tumor (FMT) specimens, and the IHC expression level of SPHK1 was associated with the histological grade of FMTs. IHC analysis of 88 FMT cases revealed that the expression level of SPHK1 was upregulated in 53 tumor tissues (60.2%) compared to adjacent mammary tissues. SPHK1 expression in FMTs was significantly associated with histological grade, presence of lymphovascular invasion, and estrogen receptor negativity. Treatment of primary FMT cells with SPHK1 inhibitors reduced cell viability, indicating that SPHK1 acts to promote FMT cell survival. These results indicate that SPHK1 may play an important role in FMTs and may be a therapeutic target in cats with FMT. CONCLUSIONS: SPHK1 over-expression in breast cancer tissues is associated with a poor prognosis in humans. SPHK1 over-expression in more aggressive FMTs provides support for a potential role of SPHK1 inhibitors for the treatment of FMTs. Targeting SPHK1 has potent cytotoxic effects in primary FMT cells. These findings suggest that further examination of the role SPHK1 plays in FMTs will pave the way for the investigation of SPHK1 inhibitors in future clinical applications.

ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis.

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.

Metabolic Studies of Tumor Cells Using [1-13 C] Pyruvate Hyperpolarized by Means of PHIP-Side Arm Hydrogenation.

The kinetics of metabolic processes can be assessed, in real time by means of MR hyperpolarized (HP) metabolites. [1-13 C]pyruvate, hyperpolarized by means of d-DNP, is, by far, the substrate most widely applied to the investigation of several pathologies characterized by deregulated glycolytic metabolic networks, including cancer. Hyperpolarization of [1-13 C]pyruvate by means of the cost effective, fast and easy to handle PHIP-SAH (para-hydrogen induced polarization-side arm hydrogenation) method opens-up a pathway for the application of HP metabolites to a wide range of cancer-related studies. Herein, we report the first application of PHIP-SAH hyperpolarized [1-13 C]pyruvate in the investigation of upregulated glycolysis in two murine breast cancer cell lines (168FARN and 4T1). The results obtained using HP pyruvate have been validated with a conventional biochemical assay and are coherent with previously-reported lactate dehydrogenase activity measured in those cells.

Catechol-o-methyltransferase genotypes are associated with progression and biological behaviour of canine mammary tumours.

The enzyme catechol-o-methyltransferase (COMT) is responsible for inactivation of catechol estrogens, which are well-recognized carcinogenic metabolites. Two single-nucleotide polymorphisms (SNPs) in canine COMT gene were previously associated with the age of onset of mammary tumours and with the clinical progression of the disease. However, no information is available regarding the impact of other known SNPs in COMT gene in canine mammary tumours. The aim of the present study is to evaluate the influence of COMT SNP in clinicopathological features and outcome of canine mammary tumours. A case series study was conducted involving 155 non-neutered bitches with mammary tumours submitted to follow-up for 24 months after surgery. Three genotypes were considered: Genotype 1 (rs853046495); Genotype 2 (rs23350589, rs23322686, rs23336579, and rs852564758); Genotype 3 (rs851328636 and rs853133060). Genotype 1 was associated to low degree of tubular differentiation. Genotype 2 was related to the development of intermediate/high-histological-grade carcinomas and to vascular invasion. Genotype 3 was associated to reduced nuclear pleomorphism and well-differentiated carcinomas. Data from the present investigation allowed the identification of COMT genetic profiles associated with pathological features of mammary tumours that constitute relevant prognostic factors. The assessment of the COMT genotypes may represent a helpful tool in the clinical management of canine mammary tumours, assisting in the selection of individualized preventive and therapeutic strategies, according to the animals' genetic profile.

Xihuang pill promotes apoptosis of Treg cells in the tumor microenvironment in 4T1 mouse breast cancer by upregulating MEKK1/SEK1/JNK1/AP-1 pathway.

OBJECTIVE: To determine the role of the MEKK1/SEK1/JNK1/AP-1 pathway in the action of Xihuang pill (XHP) in reducing regulatory T (Treg) cell numbers in the tumor microenvironment in a 4T1 mouse breast cancer model, and to clarify the anti-tumor mechanism of XHP in breast cancer. METHODS: We established a mouse 4T1 breast cancer model. Model mice were administered XHP for 2 weeks, and tumor tissues were then removed, weighed, sliced, and homogenized. Treg cells in the tumor microenvironment were isolated by magnetic cell sorting and analyzed by immunohistochemistry and flow cytometry. Treg cell apoptosis was detected by TdT-mediated dUTP nick end labeling. mRNA expression levels of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment were detected by quantitative real-time PCR and their protein expression levels were detected by immunofluorescence staining and western blot. RESULTS: Tumor weights were significantly lower in the XHP groups compared with the untreated control group. The overall number of Treg cells in the tumor microenvironment decreased while the number of apoptotic Treg cells increased with increasing doses of XHP. mRNA and protein expression levels of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment increased with increasing doses of XHP. CONCLUSION: XHP might promote Treg cell apoptosis in the tumor microenvironment and further inhibit the tumor growth of 4T1 mouse breast cancer. The mechanism of XHP may be related to upregulation of gene and protein expression of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment.

Anti-inflammatory and anti-tumor effects of α-l-guluronic acid (G2013) on cancer-related inflammation in a murine breast cancer model.

Cancer-related inflammation (CRI) is associated with the malignant progression of several cancer types. Targeting these pathways is a novel promising strategy for cancer prevention and treatment. In this present study, we evaluated the efficacy of ?-l-guluronic acid (ALG), a potent anti-inflammatory agent on breast cancer-related inflammation both in vitro and in vivo conditions. Our results indicated that ALG can effectively inhibit the CRI and tumor-promoting mediators (COX-2, MMP2, MMP9, VEGF and proinflammatory cytokines) without direct toxic effects on the cells. Moreover, it was found that, ALG can effectively inhibit the tumor cell adhesion to extracellular matrix, seeding in implantation tissue, reduce accumulation of immunosuppressive and inflammatory cells in tumor-bearing mice. These findings were associated with decreased tumor growth, metastasis, angiogenesis and prolonged mice survival. In conclusion, our data provide a cellular and molecular justification for the use of nonsteroidal anti-inflammatory drugs (NSAIDs) in treating cancer and imply the potential anti-tumor activity of ALG therapy via inhibition of CRI. These findings could lead to the establishment of novel NSAID-based cancer therapy in the near future and open a new horizon for cancer treatment.

LFM-A13, a potent inhibitor of polo-like kinase, inhibits breast carcinogenesis by suppressing proliferation activity and inducing apoptosis in breast tumors of mice.

The goals of the present study were to define the anticancer activity of LFM-A13 (α-cyano-ß-hydroxy-ß-methyl-N-(2,5-dibromophenyl)-propenamide), a potent inhibitor of Polo-like kinase (PLK), in a mouse mammary cancer model induced by 7,12-dimethylbenz(a)anthracene (DMBA) in vivo and explore its anticancer mechanism(s). We also examined whether the inhibition of PLK by LFM-A13 would improve the efficiency of paclitaxel in breast cancer growth in vivo. To do this, female BALB/c mice received 1 mg of DMBA once a week for 6 weeks with oral gavage. LFM-A13 (50 mg/kg body weight) was administered intraperitoneally with DMBA administration and continued for 25 weeks. We found that LFM-A13, paclitaxel, and their combination have a significant effect on the DMBA-induced breast tumor incidence, mean tumor numbers, average tumor weight, and size. At the molecular level, the administration of LFM-A13 hindered mammary gland carcinoma development by regulating the expression of PLK1, cell cycle-regulating proteins cyclin D1, cyclin dependent kinase-4 (CDK-4), and the CDK inhibitor, p21. Moreover, LFM-A13 treatment upregulated the levels of IκB, the pro-apoptotic proteins Bax, and caspase-3, and down-regulated p53 and the antiapoptotic protein Bcl-2 in mammary tumors. The combination of LFM-A13 with paclitaxel was found to be more effective compared with either agent alone. Collectively, these results suggest that LFM-A13 has an anti-proliferative activity against breast cancer in vivo and that LFM-A13 and paclitaxel combination could be a strategy for the treatment of breast cancer.

Insulin, not glutamine dipeptide, reduces lipases expression and prevents fat wasting and weight loss in Walker 256 tumor-bearing rats.

Cachexia is the main cause of mortality in advanced cancer patients. We investigated the effects of insulin (INS) and glutamine dipeptide (GDP), isolated or associated, on cachexia and metabolic changes induced by Walker 256 tumor in rats. INS (NPH, 40 UI/kg, sc) or GDP (1.5g/kg, oral gavage) was once-daily administered during 11 days after tumor cell inoculation. GDP, INS or INS+GDP treatments did not influence the tumor growth. However, INS and INS+GDP prevented retroperitoneal fat wasting and body weight loss of tumor-bearing rats. In consistency, INS and INS+GDP prevented the increased expression of triacylglycerol lipase (ATGL) and hormone sensitive lipase (HSL), without changing the expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in the retroperitoneal adipose tissue of tumor-bearing rats. INS and INS+GDP also prevented anorexia and hyperlactatemia of tumor-bearing rats. However, INS and INS+GDP accentuated the loss of muscle mass (gastrocnemius, soleus and long digital extensor) without affecting the myostatin expression in the gastrocnemius muscle and blood corticosterone. GDP treatment did not promote beneficial effects. It can be concluded that treatment with INS (INS or INS+GDP), not with GDP, prevented fat wasting and weight loss in tumor-bearing rats without reducing tumor growth. These effects might be attributed to the reduction of lipases expression (ATGL and LHS) and increased food intake. The results show the physiological function of INS in the suppression of lipolysis induced by cachexia mediators in tumor-bearing rats.

Congenic rats with higher arylamine N-acetyltransferase 2 activity exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism.

BACKGROUND: Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2 rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility. METHODS: Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats. RESULTS: Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01). CONCLUSIONS: A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

Molecular Characterization and In-Silico Analysis of the Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) Gene of Canine Mammary Tumor.

BACKGROUND: Mammary tumors are the second most common tumors (after skin tumors) in female dogs (Canis lupus familiaris). Tissue Inhibitor of Metlloproteinases-3 (TIMP-3) is a matrix associated endogenous inhibitor of Matrix Metalloproteinases (MMPs). Cancer metastasis occurs as a result of imbalance between MMPs and TIMPs. TIMP-3 is involved significantly in regulation of MMPs as well as progression of canine mammary tumor. OBJECTIVE: The present study was conducted to identify the structural and functional relationship between TIMP-3 and MMP which can aid in identifying the role of these proteins in canine mammary tumor. METHODS: Molecular characterization of TIMP-3 protein was done by molecular biology techniques such as gene cloning and sequencing. The homology based model of TIMP-3 protein was created and verified with a variety of available computational techniques as well as molecular dynamics simulation. RESULTS: The results indicated that predicted TIMP-3 protein structure of Canis lupus familiaris was reliable and more stable. The docking of TIMP-3 protein with MMP-2 and MMP-9 represents conformational structure of these two proteins which interact with each other but if misled canresult in the progression of tumor in canine. CONCLUSIONS: The three dimensional structure of TIMP-3 was generated and its interactions with MMP-2 and MMP-9, demonstrates the role of key binding residues. Until now, no structural details were available for canine TIMP-3 proteins, hence this study will broaden the horizon towards understanding the structural and functional aspects of this proteins in canine.

A first immunohistochemistry study of transketolase and transketolase-like 1 expression in canine hyperplastic and neoplastic mammary lesions.

BACKGROUND: Canine mammary tumors represent the most common neoplasm in female dogs, and the discovery of cancer biomarkers and their translation to clinical relevant assays is a key requirement in the war on cancer. Since the description of the 'Warburg effect', the reprogramming of metabolic pathways is considered a hallmark of pathological changes in cancer cells. In this study, we investigate the expression of two cancer-related metabolic enzymes, transketolase (TKT) and transketolase-like 1 (TKTL1), involved in the pentose phosphate pathway (PPP), an alternative metabolic pathway for glucose breakdown that could promote cancer by providing the precursors and energy required for rapidly growing cells. RESULTS: TKT and TKTL1 protein expression was investigated by immunohistochemistry in canine normal (N = 6) and hyperplastic glands (N = 3), as well as in benign (N = 11) and malignant mammary tumors (N = 17). TKT expression was higher in hyperplastic lesions and in both benign and malignant tumors compared to the normal mammary gland, while TKTL1 levels were remarkably higher in hyperplastic lesions, simple adenomas and simple carcinomas than in the normal mammary glands (P < 0.05). CONCLUSIONS: This study reveals that the expression of a key PPP enzyme varies along the evolution of canine mammary neoplastic lesions, and supports a role of metabolic changes in the development of canine mammary tumors.

RhoA GTPase oxidation stimulates cell proliferation via nuclear factor-κB activation.

Reactive oxygen species (ROS) produced by many kinds of stimuli are essential for cellular signaling including cell proliferation. The dysregulation of ROS, therefore, is related to a variety of diseases including cancer. However, it was not clearly elucidated how ROS regulate cell proliferation and tumorigenesis. In this study, we investigated a mechanism by which the oxidation of RhoA GTPase regulates nuclear factor-κB (NF-κB) and cell proliferation. Hydrogen peroxide activated NF-κB and RhoA GTPase, but did not activate RhoA C16/20A mutant, an oxidation-resistant form. Remarkably, the oxidation of RhoA reduced its affinity towards RhoGDI, leading to the dissociation of RhoA-RhoGDI complex. Si-Vav2, a guanine nucleotide exchange factor (GEF), inhibited RhoA activation upon hydrogen peroxide. The oxidized RhoA (oxRhoA)-GTP was readily bound to IκB kinase γ (IKKγ), whereas oxidized RhoGDI did not bind to IKKγ. The oxRhoA-GTP bound to IKKγ activated IKKß, leading to IκB phosphorylation and degradation, consequently NF-κB activation. Hydrogen peroxide induced cell proliferation, but RhoA C16/20A mutant suppressed cell proliferation and tumorigenesis. Conclusively, RhoA oxidation at Cys16/20 is critically involved in cell proliferation and tumorigenesis through NF-κB activation in response to ROS.

Analysis of Obesity-Related Factors and their Association with Aromatase Expression in Canine Malignant Mammary Tumours.

This study was designed to investigate the role of obesity in canine malignant mammary tumours (CMMTs), by assessing aromatase expression and the regulatory roles of immune mediators such as cyclo-oxygenase-2 (COX2), prostaglandin E2 (PGE2), nuclear factor kappa beta (NF-κB), hypoxia inducible factor-1α (HIF-1α) and adipokines (i.e. leptin) in lean, optimal body weight, overweight and obese animals. Clinicopathological data, including the breed, body weight, body condition score and age and neutering status, were collected, together with histopathological characteristics (i.e. histological types, grading and lymphatic invasion). To determine the expression of each factor, immunohistochemistry was conducted with 60 samples of malignant CMMTs. CMMTs from overweight and obese animals had significantly elevated levels of PGE2, and aromatase expression correlated significantly with PGE2, NF-κB and leptin expression. However, no significant difference was observed in terms of histopathological characteristics. The results suggest that PGE2, a known obesity-related immune mediator, could be upregulated in CMMTs from overweight and obese animals. In addition, PGE2, NF-κB and leptin influenced the expression of aromatase, as observed in women.