Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia,HIF1 induction and NFκB activation in the tumor stromal microenvironment

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB—another inducer of autophagy—prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (−/−) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the “autophagic tumor stroma model of cancer metabolism”, and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a “lethal” tumor microenvironment.Key words: caveolin-1, autophagy, BNIP3, cancer-associated fibroblasts, HIF1, hypoxia, LC3, mitophagy, NFκB, oxidative stress, predictive biomarker, TIGAR, tumor stroma     

HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts,and as a tumor suppressor in breast cancer cells: Autophagy drives compartment-specific oncogenesis

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1α-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1α-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1α in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1α showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1α also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1α increased tumor mass by ∼2-fold and tumor volume by ∼3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1α also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1α is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1α expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1α in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1α in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a three-fold reduction in tumor volume. We conclude that HIF1α activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly “feed” cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their “self-digestion.” Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other “classic” oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed “autophagic tumor stroma model of cancer.”Key words: caveolin-1, autophagy, mitophagy, the Warburg effect, tumor stroma, hypoxia, HIF1A, NFκB, compartment-specific oncogenesis, cancer-associated fibroblasts     

Phenotypical characteristics of Shiga-like toxin Escherichia coli isolated from sheep dairy products

AIMS: To analyse phenotypical characteristics of Shiga toxin-producing Escherichia coli (STEC) strains from ovine origin. METHODS AND RESULTS: A total of 13 STEC strains (eight O157 and five non-O157) isolated from sheep dairy products were used in this study. Biochemical traits, motility, haemolytic activity, resistance to tellurite-cefixime, maximum growth temperature and antibiotic resistance were determined. The STEC strains were grouped into nine biochemical and physiological biotypes (five for the O157 and four for the non-O157 strains). All STEC strains showed resistance to bacitracin, cloxacilin, penicillin and tylosin. CONCLUSIONS: Different biotypes and antibiotic resistance patterns of STEC isolated from sheep dairy products were observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will be a contribution to the better characterization of STEC isolated from sheep dairy products, which have, to date, been scarcely studied, and to the better understanding of the risks associated with its consumption.     

Non-photoperiodic factors and timing of breeding in blue tits: impact of environmental and social influences in semi-natural conditions

The seasonal development of life-history traits is influenced by many environmental factors. The impact of photoperiodic and non-photoperiodic factors on nest building and egg laying has been rarely investigated in non-domesticated avian species for which long term field data sets are available. Former investigations showed that blue tits originating from geographically close populations in the Mediterranean region do not respond in the same way to photoperiodic factors in semi-natural outdoor conditions. Here we show experimentally that nest building and onset of egg laying in captive blue tits is also proximately influenced by non-photoperiodic factors, including aspects related to aviary characteristics and social interactions between birds of the two sexes originating from different local Mediterranean study populations. In two successive experiments, we show that (1) increasing the volume of the aviary advanced the egg laying period of one specific population by almost 1 month, and (2) crossing pairs of birds from different origins strongly reduced the nest building and egg laying behaviours. These results indicate that obtaining biologically relevant breeding results in captivity with wild birds requires the control and experimental manipulation of a wide array of complex environmental cues.     

ECRG4 Expression in Normal Rat Tissues: Expression Study and Literature Review

The Esophageal Cancer Related Gene 4 (ECRG4) is a highly conserved tumour suppressor gene encoding various peptides (augurin, CΔ16 augurin, ecilin, argilin, CΔ16 argilin) which can be processed and secreted. In the present work, we examined ECRG4 expression and location in a wide range of rat organs and reviewed the available literature. ECRG4 mRNA was identified in all examined tissues by quantitative PCR (qPCR). ECRG4 immunoreaction was mainly cytoplasmic, and was detected in heart and skeletal muscles, smooth muscle cells showing only weak reactions. In the digestive system, ECRG4 immunostaining was stronger in the esophageal epithelium, bases of gastric glands, hepatocytes and pancreatic acinar epithelium. In the lymphatic system, immunoreactive cells were detectable in the thymus cortex, lymph node medulla and splenic red pulp. In the central and peripheral nervous systems, different neuronal groups showed different reaction intensities. In the endocrine system, ECRG4 immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei, hypophysis, thyroid and parathyroid glands, adrenal zona glomerularis and medulla and Leydig cells, as well as in follicular and luteal cells of the ovary. In the literature, ECRG4 has been reported to inhibit cell proliferation and increase apoptosis in various cell types. It is down-regulated, frequently due to hypermethylation, in esophageal, prostate, breast and colon cancers, together with glioma (oncosuppressor function), although it is up-regulated in papillary thyroid cancer (oncogenic role). ECRG4 expression is also higher in non-proliferating cells of the lymphatic system. In conclusion, our identification of ECRG4 in many structures suggests the involvement of ECRG4 in the tumorigenesis of other organs and also the need for further research. In addition, on the basis of the location of ECRG4 in neurons and endocrine cells and the fact that it can be secreted, its role as a neurotransmitter/neuromodulator and endocrine factor must be examined in depth in the future.Key words: Augurin, gene expression, immunohistochemistry, rat, ECRG4     

KISS1 and KISS1R expression in the human and rat carotid body and superior cervical ganglion

KISS1 and its receptor, KISS1R, have both been found to be expressed in central nervous system, but few data are present in the literature about their distribution in peripheral nervous structures. Thus, the aim of the present study was to investigate, through immunohistochemistry, the expression and distribution of KISS1 and KISS1R in the rat and human carotid bodies and superior cervical ganglia, also with particular reference to the different cellular populations. Materials consisted of carotid bodies and superior cervical ganglia were obtained at autopsy from 10 adult subjects and sampled from 10 adult Sprague-Dawley rats. Immunohistochemistry revealed diffuse expression of KISS1 and KISS1R in type I cells of both human and rat carotid bodies, whereas type II cells were negative. In both human and rat superior cervical ganglia positive anti-KISS1 and -KISS1R immunostainings were also selectively found in ganglion cells, satellite cells being negative. Endothelial cells also showed moderate immunostaining for both KISS1 and KISS1R. The expression of both kisspeptins and kisspeptin receptors in glomic type I cells and sympathetic ganglion cells supports a modulatory role of KISS1 on peripheral chemoreception and sympathetic function. Moreover, local changes in blood flow have been considered to be involved in carotid body chemoreceptor discharge and kisspeptins and kisspeptin receptors have also been found in the endothelial cells. As a consequence, a possible role of kisspeptins in the regulation of carotid body blood flow and, indirectly, in chemoreceptor discharge may also be hypothesized.     

Sleeping birds do not respond to predator odour

Background

During sleep animals are relatively unresponsive and unaware of their environment, and therefore, more exposed to predation risk than alert and awake animals. This vulnerability might influence when, where and how animals sleep depending on the risk of predation perceived before going to sleep. Less clear is whether animals remain sensitive to predation cues when already asleep.

Methodology/Principal Findings

We experimentally tested whether great tits are able to detect the chemical cues of a common nocturnal predator while sleeping. We predicted that birds exposed to the scent of a mammalian predator (mustelid) twice during the night would not go into torpor (which reduces their vigilance) and hence would not reduce their body temperature as much as control birds, exposed to the scent of another mammal that does not represent a danger for the birds (rabbit). As a consequence of the higher body temperature birds exposed to the scent of a predator are predicted to have a higher resting metabolic rate (RMR) and to lose more body mass. In the experiment, all birds decreased their body temperature during the night, but we did not find any influence of the treatment on body temperature, RMR, or body mass.

Conclusions/Significance

Our results suggest that birds are not able to detect predator chemical cues while sleeping. As a consequence, antipredatory strategies taken before sleep, such as roosting sites inspection, may be crucial to cope with the vulnerability to predation risk while sleeping.     

Spontaneous and mutagen-induced loss of DNA mismatch repair in Msh2-heterozygous mammalian cells

We have developed a simple procedure that enables the efficient selection of cells that are deficient for DNA mismatch repair (MMR). This selection procedure was used to investigate the frequency of fortuitous MMR-deficient cells in a mouse embryonic stem cell line, heterozygous for the MMR gene Msh2. We found a surprisingly high frequency (3 x 10(-4)) of Msh2-deficient cells. The wild type Msh2 allele was almost invariably lost by loss of heterozygosity. Single treatments with the genotoxic agents ethylnitrosourea, UVC light and mitomycin C resulted in a further increase of the number of Msh2-/- cells in the heterozygous cell line. This increase was not only due to induced loss of the wild type allele but also to a selective growth advantage of preexisting Msh2-/- cells to ethylnitrosourea and UVC. Mitomycin C, in contrast to ethylnitrosourea and UVC, uniquely induced loss of heterozygosity at Msh2. These mechanistically different ways of loss of the wild type Msh2 allele reflect the different repair pathways processing these damages. Heterozygous germ line defects in one of the MMR genes underlie the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Based on the results described here we hypothesize that mutagen-induced loss of MMR in the intestine of these patients contributes to the tissue specificity of carcinogenesis in HNPCC patients.