PyMT-Maclow: A novel, inducible, murine model for determining the role of CD68 positive cells in breast tumor development

CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability. We hypothesized that specific and controlled macrophage depletion would provide precise data on the effects of reducing TAM numbers on tumor development. In this study, the MacLow mouse model of doxycycline-inducible and selective CD68+ macrophage depletion was crossed with the murine mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) mouse model of spontaneous ductal breast adenocarcinoma to generate the PyMT-MacLow line. In doxycycline-treated PyMT-MacLow mice, macrophage numbers were decreased in areas surrounding tumors by 43%. Reducing the number of macrophages by this level delayed tumor progression, generated less proliferative tumors, decreased the vascularization of carcinomas and down-regulated the expression of many pro-angiogenic genes. These results demonstrate that depleting CD68+ macrophages in an inducible and selective manner delays the development of mammary tumors and that the PyMT-MacLow model is a useful and unique tool for studying the role of TAMs in breast cancer

Mitochondrial permeabilization engages NF-κB-dependent anti-tumour activity under caspase deficiency

Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies

cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation.

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I⁻/⁻). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I⁻/⁻, suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation

Breast cancer dependence on MCL-1 is due to its canonical anti-apoptotic function-AAM

High levels of the anti-apoptotic BCL-2 family member MCL-1 are frequently found in breast cancer and, appropriately, BH3-mimetic drugs that specifically target MCL-1’s function in apoptosis are in development as anti-cancer therapy. MCL-1 also has reported non-canonical roles that may be relevant in its tumour-promoting effect. Here we investigate the role of MCL-1 in clinically relevant breast cancer models and address whether the canonical role of MCL-1 in apoptosis, which can be targeted using BH3-mimetic drugs, is the major function for MCL-1 in breast cancer. We show that MCL-1 is essential in established tumours with genetic deletion inducing tumour regression and inhibition with the MCL-1-specific BH3-mimetic drug S63845 significantly impeding tumour growth. Importantly, we found that the anti-tumour functions achieved by MCL-1 deletion or inhibition were completely dependent on pro-apoptotic BAX/BAK. Interestingly, we find that MCL-1 is also critical for stem cell activity in human breast cancer cells and high MCL1 expression correlates with stemness markers in tumours. This strongly supports the idea that the key function of MCL-1 in breast cancer is through its anti-apoptotic function. This has important implications for the future use of MCL-1-specific BH3-mimetic drugs in breast cancer treatment

Differential requirements for MDM2 E3 activity during embryogenesis and in adult mice

The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion

A Unique Panel of Patient-Derived Cutaneous Squamous Cell Carcinoma Cell Lines Provides a Preclinical Pathway for Therapeutic Testing

Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation

Formate induces a metabolic switch in nucleotide and energy metabolism

Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism

Impact of formate supplementation on body weight and plasma amino acids

Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here, we investigated the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM of sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology analyser. At the end of the study, blood and tissues were collected for metabolomics analysis and immune cell profiling. Formate supplementation had no significant physiological effect on male mice, except for a small decrease in body weight. Formate supplementation had no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice, however, the body weight and spleen wet weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of bifidobacteria, a probiotic bacterium, in the stools of female mice. We conclude that formate supplementation induces physiological changes in a gender-specific manner

The MSP-RON axis stimulates cancer cell growth in models of triple negative breast cancer

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage‐stimulating protein (MSP) and its tyrosine kinase receptor, RON, are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1 . After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14‐Cre;Brca1 F/F;Trp53 F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates AKT and ERK1/2 activation as well as cancer cell growth in cell lines derived from the two mouse models, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki‐67. Analysis of human gene expression data confirmed that the genes encoding MSP and RON are robustly expressed in human TNBC as well as other subsets of breast cancer. Our findings uncover a mouse model where MSP and RON expression are naturally increased, and they provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of breast cancer