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International audience; 1 MMP11 overexpression is a bad prognostic factor in various human carcinomas. Interestingly, this proteinase is not expressed in malignant cells themselves but is secreted by adjacent non-malignant mesenchymal/stromal cells, such as cancer associated fibroblasts (CAFs) and adipocytes (CAAs), which favors cancer cell survival and progression. As MMP11 negatively regulates adipogenesis in vitro, we hypothesized that it may play a role in whole body metabolism and energy homeostasis. We used an in vivo gain-(Mmp11-Tg mice) and loss-(Mmp11−/− mice) of-function approach to address the systemic function of MMP11. Strikingly, MMP11 overexpression protects against type 2 diabetes while Mmp11−/− mice exhibit hallmarks of metabolic syndrome. Moreover, Mmp11-Tg mice were protected from diet-induced obesity and display mitochondrial dysfunction, due to oxidative stress, and metabolic switch from oxidative phosphorylation to aerobic glycolysis. This Warburg-like effect observed in adipose tissues might provide a rationale for the deleterious impact of CAA-secreted MMP11, favouring tumor progression. MMP11 overexpression also leads to increased circulating IGF1 levels and the activation of the IGF1/AKT/FOXO1 cascade, an important metabolic signalling pathway. Our data reveal a major role for MMP11 in controlling energy metabolism, and provide new clues for understanding the relationship between metabolism, cancer progression and patient outcome. More and more epidemiological data attribute a pernicious effect to metabolic disorders, notably obesity and diabetes , on cancer progression. Moreover, metformin, a drug commonly administered for type 2 diabetes, displays antitumor properties 1. To date, most studies have emphasized tumor aggressiveness in a context of already constituted metabolic disorders. Less attention has been given to the ability of cancer cells to impact on metabolism via molecules abnormally expressed by primary tumors and/or metastases. This effect may be of interest and should therefore be evaluated to unravel the link between metabolism and cancer. Matrix metalloproteinase 11 (MMP11; previously named stromelysin-3) 2

Cyclooxygenase-2 (Cox-2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox-2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox-2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox-2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox-2 gene [also known as prostaglandin-endoperoxide synthase 2 (Ptgs2)] and a Cox-2 selective drug celecoxib. Our results show that genetic deletion of Cox-2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox-2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8-14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox-2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox-2 is sufficient to reversibly disrupt gastric adenomas in mice.

While the participation of adipocytes is well known in tissue architecture, energy supply and endocrine processes, their implication during natural cancer history is just beginning to unfold. An extensive review of the literature concerning the impact of resident adipocytes on breast cancer development/progression was performed. This review provides in vitro and in vivo evidence that adipocytes located close to invasive cancer cells, referred to as cancer-associated adipocytes (CAAs), are essential for breast tumor development/progression. Their deleterious function is dependent, at least partly, on their crosstalk with invasive cancer cells. Indeed, this event leads to dramatic phenotypic and/or functional modifications of both cell types. Adipocytes exhibit delipidation and acquire a fibroblast-like shape. In parallel, cancer cell aggressiveness is exacerbated through increased migratory and invasive properties. Moreover, obesity is currently a sign of poor prognosis in human carcinomas. In this context, a high number of "obese" resident adipocytes might be predicted to be detrimental. Accordingly, there are some similarities between the molecular alterations observed in hypertrophied adipocytes and in CAAs. How adipocytes function to favor tumorigenesis at the molecular level remains largely unknown. Nevertheless, progress has been made recently and molecular clues are starting to emerge. Deciphering the cellular and molecular mechanisms behind the adipocyte-cancer cell heterotypic crosstalk is of great interest since it might provide new targets for improving diagnosis/prognosis and for the design of innovative therapeutic strategies. They might also improve our understanding of the relationship between obesity/metabolic disorders and cancer risk and/or poor patient outcome.

Interactions between pregnancy and breast cancer are complex and paradoxical. Epidemiological data show that nulliparity and late full-term pregnancy increase breast cancer risk. By contrast, early full-term pregnancy and multiparity are thought to be the most effective means of decreasing lifetime breast cancer risk. Paradoxically, young women diagnosed with breast cancer during pregnancy have a higher risk of dying from their disease. Moreover, there is a transient increase in risk of breast cancer in the first three to four years after pregnancy. After breast cancer treatment, there is no evidence that pregnancy increases the risk of breast cancer recurrence. Thus, it is not contraindicated in women previously treated for breast cancer and free of recurrence. Various physio-pathological mechanisms are involved in the protective effect of pregnancy, like cellular differentiation of mammary cells, mammary gland involution, circulating anti-mucin antibody and excretion in the milk of breast carcinogens. In the past, unfavorable effects of pregnancy were mainly attributed to precancerous cell proliferation induced by pregnancy-associated hormonal changes. However, recent studies suggest that the remodeling of cellular microenvironment and extracellular matrix during pregnancy and involution may contribute to enhanced invasive and metastatic potential of breast carcinomas.

pS2/TFF1 overexpression in breast carcinomas correlates with response to hormonotherapy. We evaluated the clinical relevance of urinary pS2/TFF1 in breast cancer patients. In healthy controls (100 cases), it represents an individual and relatively stable parameter. Although 24 out 83 pre-operative breast cancer patients showed elevated levels, both the sensitivity and specificity of the test were too low for breast cancer screening. However, neoadjuvant hormonotherapy decreased pS2/TFF1 levels in nine out of 20 patients. Furthermore, among 22 patients receiving long-term adjuvant hormonotherapy, four exhibited elevated levels, two of them at the time of relapse. Thus, urinary pS2/TFF1 quantification might be suitable as an in vivo diagnosis for tumor hormonodependency, and disease follow-up during hormonotherapy.

International audience; The in vivo disposition and antitumor efficacy of a newly developed phosphinic matrix metalloproteinase inhibitor (RXP03) were examined. RXP03 potently inhibits MMP-11, MMP-8 and MMP-13, but not MMP-1 and MMP-7. Twenty-four hours after i.p. injection into mice, most of the RXP03 was recovered intact in plasma, feces (biliary excretion) and tumor tissue. Pharmacokinetic parameters indicated that, after an i.p. dose of 100 microg/day, the plasma concentration of RXP03 over 24 hr remained higher than the Ki values determined for MMP-11, MMP-8 and MMP-13. Efficacy of RXP03 on the growth of primary tumors induced by s.c. injection of C(26) colon carcinoma cells in mice was observed to depend both on RXP03 doses and treatment schedules. Tumor volumes in mice treated for 18 days with 50, 100 and 150 microg/day of RXP03 were decreased compared with control tumor volumes, 100 microg/day being the most effective dose. Treatment at higher dose (600 microg/day) did not significantly reduce the tumor size as compared to control. Short treatments with RXP03 100 microg/day, 3 to 7 days after C(26) inoculation, were more effective on tumor growth than continuous treatment over 18 days. Strikingly, RXP03 treatment started 6 days after the C(26) injection and continued until day 18 led to stimulation of tumor growth, as compared to control. These paradoxical effects, depending on the RXP03 treatment schedule, underline the need to define carefully the spatiotemporal function of each MMP at various stages of tumor growth to achieve optimal therapeutic effects by MMP inhibitor treatment.

Epidemiological studies have shown that the use of nonsteroid anti-inflammatory drugs (NSAIDs) is associated with reduced risk of gastric cancer. The best-known target of NSAIDs is the cyclooxygenase (Cox) enzyme. Two Cox genes have been cloned, of which Cox-2 has been connected with gastric carcinogenesis. Expression of Cox-2 is elevated in gastric adenocarcinomas, which correlates with several clinicopathological parameters, including depth of invasion and lymph node metastasis. This suggests that Cox-2-derived prostanoids promote aggressive behavior of adenocarcinomas of the stomach. Cox-2 expression is especially prominent in intestinal-type gastric carcinoma and it is already present in dysplastic precursor lesions of this disease, which suggests that Cox-2 contributes to gastric carcinogenesis already at the preinvasive stage. Our most recent data show that Cox-2 is expressed in gastric adenomas of trefoil factor 1 deficient mice. Treatment of these mice with a Cox-2 selective inhibitor, celecoxib, reduced the size of the adenomas. Taken together these data support efforts to initiate clinical studies to investigate the effect of Cox-2 inhibitors as chemotherapeutic agents and as adjuvant treatment modalities against gastric neoplasias.

A novel protein expressed by entero-endocrine cells of the mouse stomach was named prepromotilin Related Peptide (ppMTLRP) since it shares sequence similarities with the prepromotilin (Tomasetto et al.). The mouse ppMTLRP was found identical to the rat precursor of ghrelin (ppghrelin), an endogenous ligand specific for the Growth Hormone Secretagogue receptor identified from rat stomach (Kojima et al.). In the present study the cDNA encoding the dog counterpart of ppMTLRP/Ghrelin has been isolated and sequenced. The dog ppMTLRP/Ghrelin cDNA showed scores of respectively 80% and 75% homology with its human and mouse counterparts. By translation of the dog ppMTLRP/Ghrelin cDNA sequences, two ORFs could be deduced encoding either a 117 amino acid ppMTLRP/Ghrelin or the deleted Gln14 ppMTLRP/Ghrelin, as it was also known in mouse, rat and man. The dog ppMTLRP/Ghrelin shared 91% similarity and 78% identity, and 89% similarity and 78% identity with the human and mouse ppMTLRP/Ghrelin proteins respectively. The best score of homology was found in the MTLRP/Ghrelin sequence itself. Indeed the dog MTLRP/Ghrelin peptide shared 100% similarity and 93% identity, and 96% identity and similarity, with the human and mouse MTLRP/Ghrelin. Using Northern blot analysis to study dog ppMTLRP/Ghrelin gene expression on dog adult gut tissues, maximal expression level was found in the stomach fundus and corpus, and no expression could be detected in the stomach antrum nor in the duodenum, jejunum, ileum, colon or liver. In conclusion, we have identified ppMTLRP/Ghrelin from dog, and found that it is highly conserved with man, mouse or rat. The expression pattern along the gastro-intestinal tract is similar to the expression pattern previously described in mouse.

Ghrelin (Ghr), a 28 amino acid gastric peptide with an n-octanoylation on Ser 3, has recently been identified as an endogenous ligand of the growth hormone secretagogue (GHS) receptor. A cDNA was also isolated from a mouse stomach library encoding a protein named prepromotilin-related peptide (ppMTLRP) which shares sequence similarities with prepromotilin. Mouse and rat ppMTLRP sequences (rGhr) are identical and show 89% identity with human ghrelin (hGhr). By analogy with promotilin, cleavage of proMTLRP into an 18 amino acid endogenous processed peptide can be assumed on the basis of a conserved dibasic motif in position 9–10 of its sequence. In the present work, we compared the GH-releasing activity of rGhr28/MTLRP and of hGhr28/MTRLP with that of a shorter form of the peptide, hGhr18. A short peptide devoid of Ser-3 n-octanoylation hGhr18[–] was also tested. Addition of rGhr28, hGhr28 and hGhr18 stimulated GH release to the same extent from superfused pituitaries. The effect was dose dependent in a 10–8 to 10–6M concentration range. In contrast, hGhr 18[–] was inactive. In freely moving animals, both rGhr28 and hGhr28 (10 µg, i.v.) stimulated GH release, whereas the same dose of hGhr18 or of hGhr18[–] was ineffective. After rGhr28, GH plasma levels increased as early as 5 min after injection and returned to basal values within 40–60 min. Expressed as percent stimulation, administration of rGhr28 was equally effective when injected during troughs or peaks of GH. Plasma concentrations of prolactin, adrenocorticotropin and leptin were not modified. Spontaneous GH secretory episodes were no longer observed within 3 h of rGhr28 treatment, but repeated administration of the secretagogue at 3- to 4-hour intervals resulted in a similar GH response. Activation of somatostatin (SRIH) release by ether stress did not blunt the GH response to rGhr28. This suggests that the secretagogue acts in part by inhibiting endogenous SRIH, as further substantiated by the ability of rGhr28 (10–6M), to decrease the amplitude of 25 mM K+-induced SRIH release from perifused hypothalami. In conclusion, (1) n-octanoylation of Ghrs and the shorter form hGhr18 is essential for the direct pituitary GH-releasing effect of this new family of endogenous GHSs; (2) only the longer forms are active in vivo and (3) inhibition of SRIH release appears involved in the mechanism of Ghr action.

Abstract —The hypothesis that stromelysin-3 (MMP-11), a unique member of the matrix metalloproteinase (MMP) family, plays a role in neointima formation was tested with the use of a vascular injury model in wild-type (MMP-11 +/+ ) and MMP-11–deficient (MMP-11 −/− ) mice. Neointima formation 2 to 3 weeks after electric injury of the femoral artery was significantly enhanced in MMP-11 −/− as compared with MMP-11 +/+ mice, in both mice of a pure 129SV genetic background (0.014 versus 0.0010 mm 2 at 2 weeks, P 2 at 3 weeks, P −/− as compared with MMP-11 +/+ arteries, in mice of both the 129SV (1.0 versus 0.18, P P −/− mice than in those from MMP-11 +/+ mice (210 versus 48, P P −/− mice. Degradation of the internal elastic lamina was more extensive in arteries of MMP-11 −/− mice than in those of MMP-11 +/+ mice (39% versus 6.8% at 3 weeks, P

Matrix metalloproteinase 2 (MMP2) activity is associated with the aggressiveness of human cancers. Therefore, the mechanisms regulating its activation are of great interest for a better understanding of malignant invasive processes. MT1-MMP, a membrane-bound MMP, is involved in the conversion of the latent form of MMP2 to the active one. In the present study, we have raised 3 monoclonal antibodies (Mabs) directed against 3 different epitopes of human MT1-MMP, which we used to investigate the expression and cellular localization of MT1-MMP protein in human carcinomas. MT1-MMP protein was present in all invasive carcinomas tested, and it was almost exclusively located to the stromal cells and not to cancer cells as previously reported, suggesting that MMP2 activation might be a peri-fibroblastic event.

Using in situ hybridization, human hair keratin basic 1 (hHb1) gene expression was investigated in human normal scalp. hHb1 transcripts were specifically detected in the cortical cells of hair shaft but neither in the outer and inner root sheaths, nor in the hair cuticle or the medulla. hHb1 expression was detected strongly in cortical cells located from the beginning of the keratogenous zone up to the isthmus. These data specify the localization of hHb1 expression. Furthermore, neoplasms with follicular differentiation, including trichoblastoma, trichoepithelioma, pilomatricoma, pilar carcinoma and basal-cell carcinoma, were analysed for hHb1 gene expression. One of the 4 pilomatricoma specimens examined exhibited a very high level of hHb1 transcripts. Interestingly, this labeling was specifically associated to a transitional cell layer en route to trichocytic differentiation, providing evidence that in pilomatricoma, epithelial germ cells can differentiate towards hair shaft keratinocytes before evolving in ghost cells.

Stromelysin-3 (ST3) is an extracellular proteinase predominantly expressed in fibroblasts. The particular structural features and in vitro functions of this molecule suggest it could be the first member of a new subgroup of the matrix metalloproteinase family. ST3 is transiently expressed during mammary gland post-weaning involution, embryonic implantation, various organogeneses, and during amphibian metamorphosis. Moreover, ST3 is expressed in a panel of human invasive carcinomas including breast, colon, and head and neck carcinomas. Almost all ST3-expressing tissues show intense extracellular matrix remodeling activities including the loss of basement membrane integrity. Thus, either directly, or indirectly in association with other proteinases, ST3 might be involved in tissue remodeling processes occurring in both physiological and pathological processes. In vitro and in vivo studies using malignant cells stably transfected in such a way as to modulate their ST3 expression levels indicate that ST3 modifies neither cell proliferation nor invasive properties, but rather favors tumor cell survival in host tissues. This hypothesis is consistent with clinical data showing that ST3 expression could be predictive of tumor progression leading to metastases.

This study evaluated the in vivo antitumor activity of grape-derived polyphenols. BALB/c mice were subcutaneously implanted with C26 colon carcinoma cells, and 2 d later they received either solvent or red wine polyphenols (RWPs) (100 mg/kg/d, human equivalent dose approximately 500 mg/d) in the drinking water for 25 d. Wistar rats received either solvent or RWPs (100 mg/kg/d, human equivalent dose approximately 1000 mg/d) in the drinking water 1 wk before injection of azoxymethane and were studied 10 wk later. In mice, RWPs inhibited tumor growth by 31%, reduced tumor vascularization and the number of lung metastases, decreased proliferation as indicated by down-regulation of Ki67, cyclin D1, and UHRF1, and increased apoptosis as indicated by TUNEL staining and active caspase-3 levels in tumor cells. RWPs reduced expression of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, and cyclooxygenase-2 and increased expression of tumor suppressor genes p16(INK4A), p53, and p73 in tumor cells. In rats, RWPs reduced by 49% the number of azoxymethane-induced aberrant crypt foci (preneoplastic lesions) in colon. Thus, RWPs effectively reduced the development of colon carcinoma tumors in vivo by blunting tumor vascularization and by inhibiting proliferation and promoting apoptosis of tumor cells subsequent to an up-regulation of tumor suppressor genes.

MMP11 expression is a poor prognosis factor in human carcinomas. Although it has been shown to favor primary tumor development, its role in metastatic processes remains unclear. We studied the hematogenous metastatic activity of C26 mouse colon cancer cells injected into the tail vain of wild-type or MMP11-deficient mice during 2 months. Using X-ray computed tomography to image metastasis development in recipient living mice, lung metastases were found to occur earlier and to grow faster in wild-type mice. Histological analyses of the lung, liver, kidney, adrenal gland, mammary gland, ovary and salivary gland, performed at the end of experiment, also showed lower numbers of metastases in wild-type mice, regardless of organ. Lung metastases showed similar Factor VIII-positive vascular networks regardless of the mouse MMP11 status. However, those found in MMP11-deficient mice also exhibited vessel-like structures that did not express Factor VIII, Lyve-1 and vimentin, and were not stained with PAS. Consequently, they did not correspond to vascular or lymphatic vessels or to vascular mimicry channels. Collectively, these results revealed significant spatio-temporal variability that is dependent on host MMP11 status. Furthermore, they point-out the paradoxical role of MMP11 in favoring the onset and growth of lung metastases but limiting lung foci number, and inhibiting the cancer cell dissemination to other organs. These data highlight the complexity of the metastatic process in which the same factor can play activator or repressor functions depending on the metastatic step.

We have cloned from a mouse placenta cDNA library a mouse homologue of the human stromelysin-3 (ST3) cDNA, which codes for a putative matrix metalloproteinase expressed in breast carcinomas. The ST3 protein is well conserved between humans and mice, and the pattern of ST3 gene expression is similar in both species, and shows expression in the placenta, in the uterus, and during limb bud morphogenesis. We show that the ST3 gene can also be expressed in the normal mouse mammary gland. ST3 gene expression was not detected during mammary growth, neither in virgin nor in pregnant mice, but was specifically observed during postlactating involution of the gland, an apoptotic process associated with intense extracellular matrix remodeling. ST3 transcripts were found in fibroblasts immediately surrounding degenerative ducts, suggesting that ST3 gene expression may be associated with the basement membrane dissolution, which occurs during mammary gland involution. Since the ST3 gene is also specifically expressed in fibroblastic cells surrounding invasive neoplastic cells of breast carcinomas, we suggest that ST3 is implicated in extracellular matrix remodeling processes common to mammary apoptosis and breast cancer progression.

Methyl donor deficiency (MDD) during pregnancy influences intrauterine development. Ghrelin is expressed in the stomach of fetuses and influences fetal growth, but MDD influence on gastric ghrelin is unknown. We examined the gastric ghrelin system in MDD-induced intrauterine growth retardation. By using specific markers and approaches (such as periodic acid–Schiff, bromodeoxyuridine, homocysteine, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunostaining, reverse transcription-polymerase chain reaction), we studied the gastric oxyntic mucosa cellular organization and ghrelin gene expression in the mucosa in 20-day-old fetuses and weanling pups, and plasma ghrelin concentration in weanling rat pups of dams either normally fed or deprived of choline, folate, vitamin B6, and vitamin B12 during gestation and suckling periods. MDD fetuses weighed less than controls; the weight deficit reached 57% at weaning (P < 0.001). Both at the end of gestation and at weaning, they presented with an aberrant gastric oxyntic mucosa formation with loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Ghrelin cells were abnormally located in the pit region of oxyntic glands. At weaning, plasma ghrelin levels were decreased (−28%; P < 0.001) despite unchanged mRNA expression in the stomach. This decrease was associated with lower body weight. Taken together, these data indicate that one mechanism through which MDD influences fetal programming is the remodeling of gastric cellular organization, leading to dysfunction of the ghrelin system and dramatic effects on growth.

The pS2 gene encodes for a small cysteine-rich protein, and was originally found by differential screening of a cDNA library from the human breast carcinoma cell line, MCF-7. The presence of pS2 is closely correlated with oestrogen dependence in breast carcinomas. While the function of pS2 is unknown, pS2 protein has been shown to be homologous with the gastrointestinal peptide hormone pancreatic spasmolytic polypeptide (PSP) and its human counterpart hSP, in which a 5-cysteine domain is tandemly repeated. The 5' flanking region of the pS2 gene contains an enhancer region responsive to oestrogens and to epidermal growth factor (EGF/URO). We now report that pS2 and hSP expression occurs in a wide range of endodermally-derived tissues, including the duodenum, the pancreas, and in a recently defined cell lineage associated with chronic gastrointestinal ulceration. In each case, this expression was associated with secretion of immunoreactive EGF/URO. We further show that the co-expression of pS2 and hSP in gastric surface epithelial cells is also associated with the secretion of EGF/URO in the subjacent mucous neck cells. Our results indicate that local EGF/URO secretion induces pS2 and hSP in adjacent cells, and that these molecules are then available to participate in pathophysiological responses. The finding of similar patterns of EGF/URO, hSP and pS2 expression in association with chronic damage suggests that this is a fundamental response in the healing of these tissues.

AIM: To examine the in vivo phenotype associated with hepatic metastatic lymph node 64 (MLN64) over-expression. METHODS: Recombinant-adenovirus-mediated MLN64 gene transfer was used to overexpress MLN64 in the livers of C57BL/6 mice. We measured the effects of MLN64 overexpression on hepatic cholesterol content, bile flow, biliary lipid secretion and apoptosis markers. For in vitro studies cultured CHO cells with transient MLN64 overexpression were utilized and apoptosis by TUNEL assay was measured. RESULTS: Livers from Ad.MLN64-infected mice exhibited early onset of liver damage and apoptosis. This response correlated with increases in liver cholesterol content and biliary bile acid concentration, and impaired bile flow. We investigated whether liver MLN64 expression could be modulated in a murine model of hepatic injury. We found increased hepatic MLN64 mRNA and protein levels in mice with chenodeoxycholic acid-induced liver damage. In addition, cultured CHO cells with transient MLN64 overexpression showed increased apoptosis. CONCLUSION: In summary, hepatic MLN64 over-expression induced damage and apoptosis in murine livers and altered cholesterol metabolism. Further studies are required to elucidate the relevance of these findings under physiologic and disease conditions.

International audience; Using surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF), Li et al. [Clin Chem 48(8): 1296-1304, 2002] identified 3 serum biomarkers, BC1 (4.3 kDa), BC2 (8.1 kDa) and BC3 (8.9 kDa), whose combination significantly detects breast cancer patients from non-cancer controls. This work aimed to validate these biomarkers in an independent prospective study. We screened 89 serum samples including 49 breast cancers at pT1-4N0M0 (n = 23), pT1-4N1-3M0 (n = 17) or pT1-4N0-3M1 (n = 9) stages, 13 benign breast diseases and 27 healthy women. The BC2 biomarker significance was not recovered. However, we found 2 peaks that we named BC1a (4286 Da) and BC1b (4302 Da), that could correspond to Li's BC1 since they significantly decrease in breast cancers (p < 0.00007 and p < 0.0002, respectively). Similarly, BC3a (8919 Da) and BC3b (8961 Da) are significantly increased in breast cancers (p < 0.02 and p < 0.0002, respectively) and could correspond to the Li's BC3. For each biomarker we defined stringent (no errors) and flexible (less than 10% errors) cut-off values and tested the power of the combined BC1a/BC1b/BC3a/BC3b stringent and flexible profiles to discriminate breast cancers. They identified 33% and 45% cancers, respectively. Applied to the same series, Ca 15.3 test identified 22% patients. Interestingly, in association with the BC1a/BC1b/BC3a/BC3b profiles, Ca 15.3 improved the number of detected cancers indicating that it is an independent parameter. Collectively, our data partially validate those of Li's study and confirm that the BC1 and BC3 biomarkers are helpful for breast cancer diagnosis.

International audience; Small cell transmembrane and glycosylated protein (SMAGP) was recently identified in the metastasizing rat pancreatic adenocarcinoma line BSp73ASML. SMAGP, an evolutionary conserved transmembrane protein, is expressed on lateral epithelial cell membranes. SMAGP expression was restricted to or was upregulated in several metastasizing as compared to nonmetastasizing human and rat tumor lines. In contrast to nontransformed tissue, SMAGP was mainly expressed in the cytoplasm, as has already been described for high-grade human colorectal cancer. This raised the question on the impact of SMAGP on tumor progression. To answer the question, metastasis formation was evaluated in the nonmetastasizing rat pancreatic adenocarcinoma subline BSp73AS (AS), which was stably transfected with SMAGP cDNA (AS-SMAGP). Cytoplasmic SMAGP expression promoted cell agglomeration, but inhibited tumor cell proliferation, adhesion to and migration toward vitronectin and matrigel invasion, which was accompanied by a failure of actin reorganization. AS-SMAGP clones strongly promoted metastasis formation by dislodgment of normal tissue; 82% of rats developed lymph node metastasis as compared to 22% of rats receiving AS or mock-cDNA-transfected AS cells. The incidence of lung metastasis was increased from 6% in AS to 98% in AS-SMAGP tumor-bearing rats. Thus, SMAGP strongly promotes tumor progression. This likely is due to redistribution from the plasma membrane into the cytoplasm. SMAGP redistribution does not only facilitate tumor cell detachment from neighboring cells and the extracellular matrix, but obviously contributes actively by a not yet defined mechanism to tumor cell agglomeration and capillary plugging.

The initial invasive processes during cancer development remain largely unknown. Stromelysin-3/matrix metalloproteinase 11 (ST3/MMP11) is associated with tumor invasion and poor prognosis. We present novel evidence that adipocytes present at human breast tumor invasive front are induced by cancer cells to express ST3. Using mouse syngeneic model, light and electron microscopy showed that in ST3-deficient mice but not in wild-type mice, forced cancer cell-adipocyte interaction/crosstalk results in adipocyte membrane alteration, allowing cancer cell fat infiltration and death. Thus, adipocytes are involved in initial cancer cell survival into connective tissue, and this effect is ST3 mediated. This suggested that ST3 might play a role in adipocyte metabolism. Accordingly, ST3-deficient mice exhibited fat excess and increased mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte protein 2 (aP2) adipogenic markers, indicating that, in vivo, ST3 negatively regulates fat homeostasis. Moreover, ST3-deficient mouse embryonic fibroblasts exhibited a dramatic enhanced potential to differentiate into adipocytes associated with increased PPARγ and aP2 expression, and recombinant ST3 treatment reverted their differentiation. Thus, in vitro, ST3 reduces adipocyte differentiation in an autocrine manner. High fibroblasts/adipocytes ratio is a stroma feature, and peritumoral fibroblast origin remains debated. Our results support the concept that invading cancer cells aberrantly restore the negative ST3 function on adipogenesis into proximal adipocytes/preadipocytes, leading to the accumulation/maintenance of a particular peritumoral fibroblast subpopulation. Accordingly, in human breast tumors, we observed that ST3-expressing peritumoral fibroblasts are distinct from α-smooth muscle actin-expressing myofibroblasts. This constitutes the first report of implication of a MMP in cancer cell-adipocyte interaction/crosstalk during early steps of connective tissue invasion.

International audience; Intramammary sentinel lymph node excision during breast conservative surgery was performed, in this case report, using a prototype intraoperative gamma probe. In contrast to the four axillary sentinel lymph nodes that were subnormal, the excised intramammary sentinel lymph node was massively invaded by cancer cells. Therefore this finding had profound implication for the staging of the tumor and for treatment selection. This case report illustrates that an efficient intraoperative gamma probe is useful to locate and remove intramammary sentinel lymph node in breast cancer patients treated with breast conservation.

Trefoil factors (TFFs) are protease-resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose-response curves in src-transformed MDCKts.src and PCmsrc cells (EC50=20-40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src-pS2 cells and human colonic HCT8/S11-pS2 cells established from a sporadic tumor. Furthermore, we found that TFF-mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3'-kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed by Western blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.

pS2/TFF1 trefoil factor is normally expressed in the stomach, and is found ectopically in gastrointestinal inflammatory disorders and in various carcinomas. It is involved in stomach ontogenesis and in the maintenance of the integrity of the mucosa, and may represent a pharmacological tool for prevention and healing of gastrointestinal ulcerations. In breast cancer, it can be used to select patients suitable for hormone therapy. pS2/TFF1 is a pleiotropic factor involved in mucin polymerization, cell motility, cell proliferation and/or differentiation, and possibly in the nervous system.

To determine the function of the pS2 trefoil protein, which is normally expressed in the gastric mucosa, the mouse pS2 (mpS2) gene was inactivated. The antral and pyloric gastric mucosa of mpS2-null mice was dysfunctional and exhibited severe hyperplasia and dysplasia. All homozygous mutant mice developed antropyloric adenoma, and 30 percent developed multifocal intraepithelial or intramucosal carcinomas. The small intestine was characterized by enlarged villi and an abnormal infiltrate of lymphoid cells. These results indicate that mpS2 is essential for normal differentiation of the antral and pyloric gastric mucosa and may function as a gastric-specific tumor suppressor gene.

It has long been proposed that secreted proteinases, including the matrix metalloproteinases, play an important part in tumor progression in mediating extracellular matrix remodeling. More recently, it has been suggested that extracellular proteinases also regulate growth factors and cytokines that may contribute to tumor progression.RNA in situ hybridization and immunohistochemistry were used to study the expression, in breast and other types of human carcinomas, of the stromelysin-3 (ST3) gene, which encodes a putative new member of the matrix metalloproteinase family.The ST3 gene is overexpressed in most types of human carcinomas, including breast carcinoma where ST3 RNA was detected in 95% (99 of 104) of invasive primary tumors. Both ST3 protein and RNA are detected in fibroblastic cells immediately surrounding the cancer cells, but not in the malignant cells or in stromal cells at a distance from them. The ST3 gene also is expressed in some in situ breast carcinomas, where ST3 expression correlates with the known risk of these tumors to become invasive.ST3 is the paradigm of tumor proteinases that are not expressed in the malignant cells of human carcinomas but in fibroblastic cells of tumor stroma. ST3 represents a potential new prognostic parameter to identify subpopulations of aggressive tumors, particularly to evaluate the likelihood of in situ breast carcinoma progression to invasive cancer. Furthermore, the specific expression of the ST3 gene in fibroblastic cells immediately surrounding cancer cells suggests that ST3 may be involved in tumor progression and that it represents a potential target for cancer treatment.

The recently discovered pS2 protein is expressed under estrogen control in a subset of estrogen receptor-positive breast cancers and in an estrogen-independent manner in normal stomach mucosa. The pS2 gene belongs to a family of genes encoding peptides that contain a conserved 5-cysteine domain, the P domains. Although the function of the pS2 protein is unknown, it has been suggested that it may have cell growth stimulatory activity. We report here that expression of the pS2 gene in the digestive tract, which is normally restricted to the stomach, is strongly induced by mucosal ulcerations elsewhere in the tract, most notably in Crohn's disease. pS2 gene expression is restricted to the mucosal layers adjacent to the ulcerations, in a region where a novel epidermal growth factor-secreting cell lineage was shown to be induced by mucosal ulceration. The human hSP gene, which contains a tandem duplication of the pS2 gene P domain and is coexpressed with the pS2 gene in normal stomach mucosa but not in breast cancers, is also expressed in Crohn's disease. We suggest that pS2 gene expression may provide a useful marker for mucosal ulcerations of the digestive tract.

The hormone-dependence of some human breast cancers is well recognized. However, the molecular mechanisms responsible for the growth stimulation of these cancers by oestrogens are still poorly understood. With the hope of elucidating these mechanisms, we have recently cloned and studied the structure-function relationship of the human oestrogen and progestin receptors, and also undertaken a study aimed at characterizing genes whose expression is controlled by oestrogens in hormone-dependent breast cancers. We review here our findings concerning one of these genes and its expression products, the pS2 gene. We discuss also whether a systematic determination of pS2 gene expression in breast cancer biopsies could be useful to establish a new biochemical classification of these cancers which may be useful to improve the diagnosis of hormone-dependent cancers.

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.

The expression of the estrogen-regulated breast-cancer-associated pS2 gene was examined in 75 stomach resections taken from 45 patients. The 600-base pS2 mRNA was found in all of the 47 non-neoplastic samples at varying levels: in the histologically normal group we observed a Poisson-type distribution, whereas 79% of the tissues exhibiting dysplastic features expressed high levels of transcript. Tumour samples expressed relatively lower pS2 mRNA, with only 18% having high levels and 43% with no detectable expression. These differences were not correlated to tumour grading, stage or site. No amplification or rearrangement of the pS2 gene was found. Immunohistochemical analysis of formalin-fixed paraffin sections, using a polyclonal antibody against pS2 protein, showed specific staining of both cytoplasm and membrane of epithelial cells in the neck region of antral and body glands as well as in luminal secretions. Immunoreactivity was observed in the sub-nuclear region of foveolar cells, with specialized gland and goblet cells in atrophic gastritis being negative. Heterogeneous but strong focal cytoplasmic staining was seen in tumour cells as well as in dysplastic epithelium. Two gastric cell lines, KATO III and MKN-45, derived from poorly differentiated adenocarcinomas also expressed pS2, whereas 3 other lines from well differentiated parental tumours did not. Genomic analysis revealed a BamHI polymorphism in Kato III cells and in the non-expressing MKN-28 cells. Immunostaining to pS2 protein was also demonstrated in the cytoplasm of KATO III cells, but neither these nor any of 30 tissues examined showed any positivity with a monoclonal antibody (MAb) to estrogen receptor. Our results suggest that pS2 is normally expressed in human stomach, possibly in association with secretory activity, and becomes down-regulated during malignancy.

Our laboratory has reported previously the cloning of a complementary DNA termed pS2, corresponding to a messenger RNA (mRNA) whose synthesis is induced by estrogen in the human breast cancer cells MCF-7. Examination of the possible open reading frames of this complementary DNA has led to the prediction that the pS2 protein could be a secreted polypeptide of either 58 or 63 amino acids in length. Using a rabbit antiserum prepared against a synthetic peptide corresponding to the last 31 amino acids of the putative protein, we show that a protein with the expected migration during sodium dodecyl sulfate gel electrophoresis can indeed be immunoprecipitated from either the culture medium of MCF-7 cells grown in the presence of labeled amino acids or the in vitro translation products of MCF-7 poly(A) RNA enriched in pS2 mRNA. Furthermore, in vivo and in vitro differential amino acid labeling allows us to conclude that the mature pS2 protein is probably secreted as a 58 amino acid long peptide. Finally, we show that pS2 protein synthesis is induced in MCF-7 cells by estradiol and phenol red, but not by the antiestrogen tamoxifen, in keeping with our previous results demonstrating estrogen induction of pS2 mRNA synthesis.

The ulceration-associated cell lineage (UACL) develops in the human gastrointestinal mucosa after ulceration; it grows out from the bases of adjacent crypts and ramifies in the lamina propria to form a new gland, finally giving rise to a duct by which the glandular secretion and indeed cells are carried to the surface. Using immunocytochemistry and in situ hybridization with 35S-labelled riboprobes, we have defined the pattern of trefoil peptide gene expression (pS2; human spasmolytic polypeptide, hSP), epidermal growth factor/urogastrone (EGF/URO), and the distribution of cell proliferation during the development of the UACL, as indicated by immunostaining for proliferating cell nuclear antigen (PCNA). Our studies reveal that the morphogenesis of the UACL shows a marked morphological resemblance to developing Brunner's glands; the pattern of trefoil peptide gene expression during UACL development is also very similar. However, trefoil peptide gene expression in the mature UACL complex is unique amongst gastrointestinal cells. The mature UACL shows a distinctive proliferative organization: while the early buds and glands are non-proliferative, apparently being fed by cells from the parent crypts, a definitive proliferative zone develops within the duct. This, of course, corresponds to the location of the gastric gland proliferative zone. We propose that while the UACL shows novel features, it shares its differentiation programme with Brunner's glands, but its pattern of cell renewal eventually is that of the gastric gland.