The SEMA3F-NRP1/NRP2 axis is a key factor in the acquisition of invasive traits in in situ breast ductal carcinoma.

BACKGROUND: A better understanding of ductal carcinoma in situ (DCIS) is urgently needed to identify these preinvasive lesions as distinct clinical entities. Semaphorin 3F (SEMA3F) is a soluble axonal guidance molecule, and its coreceptors Neuropilin 1 (NRP1) and NRP2 are strongly expressed in invasive epithelial BC cells. METHODS: We utilized two cell line models to represent the progression from a healthy state to the mild-aggressive or ductal carcinoma in situ (DCIS) stage and, ultimately, to invasive cell lines. Additionally, we employed in vivo models and conducted analyses on patient databases to ensure the translational relevance of our results. RESULTS: We revealed SEMA3F as a promoter of invasion during the DCIS-to-invasive ductal carcinoma transition in breast cancer (BC) through the action of NRP1 and NRP2. In epithelial cells, SEMA3F activates epithelialmesenchymal transition, whereas it promotes extracellular matrix degradation and basal membrane and myoepithelial cell layer breakdown. CONCLUSIONS: Together with our patient database data, these proof-of-concept results reveal new SEMA3F-mediated mechanisms occurring in the most common preinvasive BC lesion, DCIS, and represent potent and direct activation of its transition to invasion. Moreover, and of clinical and therapeutic relevance, the effects of SEMA3F can be blocked directly through its coreceptors, thus preventing invasion and keeping DCIS lesions in the preinvasive state.

In Vitro and In Vivo Effects of the Combination of Polypurine Reverse Hoogsteen Hairpins against HER-2 and Trastuzumab in Breast Cancer Cells.

Therapeutic oligonucleotides are powerful tools for the inhibition of potential targets involved in cancer. We describe the effect of two Polypurine Reverse Hoogsteen (PPRH) hairpins directed against the ERBB2 gene, which is overexpressed in positive HER-2 breast tumors. The inhibition of their target was analyzed by cell viability and at the mRNA and protein levels. The combination of these specific PPRHs with trastuzumab was also explored in breast cancer cell lines, both in vitro and in vivo. PPRHs designed against two intronic sequences of the ERBB2 gene decreased the viability of SKBR-3 and MDA-MB-453 breast cancer cells. The decrease in cell viability was associated with a reduction in ERBB2 mRNA and protein levels. In combination with trastuzumab, PPRHs showed a synergic effect in vitro and reduced tumor growth in vivo. These results represent the preclinical proof of concept of PPRHs as a therapeutic tool for breast cancer.

Cancer-Associated Fibroblasts in Breast Cancer Treatment Response and Metastasis.

Breast cancer (BrCa) is the leading cause of death among women worldwide, with about one million new cases diagnosed each year. In spite of the improvements in diagnosis, early detection and treatment, there is still a high incidence of mortality and failure to respond to current therapies. With the use of several well-established biomarkers, such as hormone receptors and human epidermal growth factor receptor-2 (HER2), as well as genetic analysis, BrCa patients can be categorized into multiple subgroups: Luminal A, Luminal B, HER2-enriched, and Basal-like, with specific treatment strategies. Although chemotherapy and targeted therapies have greatly improved the survival of patients with BrCa, there is still a large number of patients who relapse or who fail to respond. The role of the tumor microenvironment in BrCa progression is becoming increasingly understood. Cancer-associated fibroblasts (CAFs) are the principal population of stromal cells in breast tumors. In this review, we discuss the current understanding of CAFs' role in altering the tumor response to therapeutic agents as well as in fostering metastasis in BrCa. In addition, we also review the available CAFs-directed molecular therapies and their potential implications for BrCa management.

Breast Mammographic Density: Stromal Implications on Breast Cancer Detection and Therapy.

Current evidences state clear that both normal development of breast tissue as well as its malignant progression need many-sided local and systemic communications between epithelial cells and stromal components. During development, the stroma, through remarkably regulated contextual signals, affects the fate of the different mammary cells regarding their specification and differentiation. Likewise, the stroma can generate tumour environments that facilitate the neoplastic growth of the breast carcinoma. Mammographic density has been described as a risk factor in the development of breast cancer and is ascribed to modifications in the composition of breast tissue, including both stromal and glandular compartments. Thus, stroma composition can dramatically affect the progression of breast cancer but also its early detection since it is mainly responsible for the differences in mammographic density among individuals. This review highlights both the pathological and biological evidences for a pivotal role of the breast stroma in mammographic density, with particular emphasis on dense and malignant stromas, their clinical meaning and potential therapeutic implications for breast cancer patients.

Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis.

BACKGROUND: The microenvironment and stress factors like glucocorticoids have a strong influence on breast cancer progression but their role in the first stages of breast cancer and, particularly, in myoepithelial cell regulation remains unclear. Consequently, we investigated the role of glucocorticoids in ductal carcinoma in situ (DCIS) in breast cancer, focusing specially on myoepithelial cells. METHODS: To clarify the role of glucocorticoids at breast cancer onset, we evaluated the effects of cortisol and corticosterone on epithelial and myoepithelial cells using 2D and 3D in vitro and in vivo approaches and human samples. RESULTS: Glucocorticoids induce a reduction in laminin levels and favour the disruption of the basement membrane by promotion of myoepithelial cell apoptosis in vitro. In an in vivo stress murine model, increased corticosterone levels fostered the transition from DCIS to invasive ductal carcinoma (IDC) via myoepithelial cell apoptosis and disappearance of the basement membrane. RU486 is able to partially block the effects of cortisol in vitro and in vivo. We found that myoepithelial cell apoptosis is more frequent in patients with DCIS+IDC than in patients with DCIS. CONCLUSIONS: Our findings show that physiological stress, through increased glucocorticoid blood levels, promotes the transition from DCIS to IDC, particularly by inducing myoepithelial cell apoptosis. Since this would be a prerequisite for invasive features in patients with DCIS breast cancer, its clinical management could help to prevent breast cancer progression to IDC.

Histamine receptor 1 inhibition enhances antitumor therapeutic responses through extracellular signal-regulated kinase (ERK) activation in breast cancer.

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine triggers cell proliferation, embryonic development, and tumor growth. We recently established that HRH1 is up-regulated in basal and human epidermal growth factor receptor 2 (HER2)-enriched human breast tumors and that its expression correlates with a worse prognosis. Nevertheless, the functional role of HRH1 in basal and HER2-targeted therapy-resistant breast cancer (BC) progression has not yet been addressed. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that the inhibition of HRH1 activity in basal BC cells leads to sub-G0 cell accumulation, suppresses proliferation, promotes cell motility and triggers the activation of extracellular signal-regulated kinase (ERK) signaling, initiating the mitochondrial apoptotic pathway. Furthermore, HER2-targeted therapy-resistant cells express higher levels of HRH1 and are more sensitive to terfenadine treatment. Moreover, in vivo experiments showed that terfenadine therapy reduced the tumor growth of basal and trastuzumab-resistant BC cells. In conclusion, our results suggest that targeting HRH1 is a promising new clinical approach to consider that could enhance the effectiveness of current therapeutic treatment in patients with basal and BC tumors resistant to HER2-targeted therapies.

Characterization of human pancreatic orthotopic tumor xenografts suitable for drug screening.

BACKGROUND: Efforts to identify novel therapeutic options for human pancreatic ductal adenocarcinoma (PDAC) have failed to result in a clear improvement in patient survival to date. Pancreatic cancer requires efficient therapies that must be designed and assayed in preclinical models with improved predictor ability. Among the available preclinical models, the orthotopic approach fits with this expectation, but its use is still occasional. METHODS: An in vivo platform of 11 orthotopic tumor xenografts has been generated by direct implantation of fresh surgical material. In addition, a frozen tumorgraft bank has been created, ensuring future model recovery and tumor tissue availability. RESULTS: Tissue microarray studies allow showing a high degree of original histology preservation and maintenance of protein expression patterns through passages. The models display stable growth kinetics and characteristic metastatic behavior. Moreover, the molecular diversity may facilitate the identification of tumor subtypes and comparison of drug responses that complement or confirm information obtained with other preclinical models. CONCLUSIONS: This panel represents a useful preclinical tool for testing new agents and treatment protocols and for further exploration of the biological basis of drug responses.

Differential regulation of MMP7 in colon cancer cells resistant and sensitive to oxaliplatin-induced cell death.

BACKGROUND: We have previously shown that metalloproteinase 7 (MMP7) expression is increased during the acquisition of resistance to oxaliplatin in colon cancer cells. Now we have analyzed the implication of ß-catenin and EGFR pathways in the up-regulation of MMP7 in the oxaliplatin-resistant human colon cancer cell lines RHT29 and RHCT116 p53-/-, derived from the HT29 and HCT116 p53-/- cells, respectively. RESULTS: Oxaliplatin treatment increased EGFR expression and induced its activation in all the cell lines. However, ß-catenin mRNA was only upregulated in the HT29 and RHT29 cells, with a marked increase in the nuclear/cytoplasmic ß-catenin protein ratio in the oxaliplatin-resistant RHT29 cells. To determine the contribution of ß-catenin and EGFR to the expression of MMP7 we performed siRNA experiments. ß-catenin abrogation only prevented the induction of MMP7 by oxaliplatin in HT29 and RHT29 cells. Accordingly, viability of oxaliplatin-treated RHT29 cells under ß-catenin silencing was decreased. On the other hand, EGFR siRNA induced contradictory effects, decreasing PEA3 and MMP7 expression in control and oxaliplatin-treated RHCT116 p53-/- cells but increasing basal- and oxaliplatin-induced PEA3 and MMP7 in the HT29 and RHT29 cells. CONCLUSIONS: Oxaliplatin-induced MMP7 up-regulation is differentially achieved in colon cancer cell lines, as a result of EGFR and ß-catenin cross-talk on MMP7 gene transcription. Taken together, our results point out the disparity of effects that ß-catenin and EGFR blocking therapeutic strategies may exert on MMP7 expression depending on the cellular context and remark the importance of a better knowledge of MMP7 regulation to improve chemotherapy effectiveness in colon cancer.

Tumor promoting effects of CD95 signaling in chemoresistant cells.

BACKGROUND: CD95 is a death receptor controlling not only apoptotic pathways but also activating mechanisms promoting tumor growth. During the acquisition of chemoresistance to oxaliplatin there is a progressive loss of CD95 expression in colon cancer cells and a decreased ability of this receptor to induce cell death. The aim of this study was to characterize some key cellular responses controlled by CD95 signaling in oxaliplatin-resistant colon cancer cells. RESULTS: We show that CD95 triggering results in an increased metastatic ability in resistant cells. Moreover, oxaliplatin treatment itself stimulates cell migration and decreases cell adhesion through CD95 activation, since CD95 expression inhibition by siRNA blocks the promigratory effects of oxaliplatin. These promigratory effects are related to the epithelia-to-mesenchymal transition (EMT) phenomenon, as evidenced by the up-regulation of some transcription factors and mesenchymal markers both in vitro and in vivo. CONCLUSIONS: We conclude that oxaliplatin treatment in cells that have acquired resistance to oxaliplatin-induced apoptosis results in tumor-promoting effects through the activation of CD95 signaling and by inducing EMT, all these events jointly contributing to a metastatic phenotype.

Adenoviral-mediated overexpression of human equilibrative nucleoside transporter 1 (hENT1) enhances gemcitabine response in human pancreatic cancer.

Nucleoside-derived anticancer agents must be transported across the plasma membrane as a preliminary step to their conversion into active drugs. Hence, modulation of a specific nucleoside transporter may affect bioavailability and contribute significantly to sensitizing tumor cells to these anticancer agents. We have generated and functionally characterized a new recombinant adenovirus (Ad-hENT1) that has allowed us to overexpress the equilibrative nucleoside transporter hENT1 and to analyze its effects in human pancreatic tumor cells. Overexpression of hENT1 is associated with changes in cell cycle profile, in a variable manner depending on the particular cell type, thus suggesting a metabolic link between hENT1-mediated transport processes and the enzymatic machinery responsible for intracellular nucleoside metabolism. When assayed in vivo in a human pancreatic adenocarcinoma xenograft, intratumoral Ad-hENT1 injection improved the therapeutic response to gemcitabine. In summary, hENT1 overexpression is associated with alterations in nucleoside enzymatic machinery and cell cycle progression in cultured cells and enhances gemcitabine action in vivo.

Resveratrol does not ameliorate muscle wasting in different types of cancer cachexia models.

BACKGROUND & AIMS: Resveratrol has been reported to have antitumoural effects and recently it has been demonstrated that resveratrol partially blocks skeletal muscle wasting by interfering with NF-kappaB activation. We decided to investigate the potential anti-wasting properties of resveratrol on different models of cancer cachexia in experimental animals. METHODS AND RESULTS: Incubations of isolated extensor digitorum longus muscles in the presence of 30 microM of resveratrol caused a significant decrease in the rate of protein degradation. However, administration of resveratrol in vivo to both rats bearing the Yoshida AH-130 ascites hepatoma (at the dose of 1 mg/kg body weight) and mice bearing the Lewis lung carcinoma (at two different doses, 5 and 25 mg/kg body weight) had no effect on skeletal muscle mass or body weight in tumour-bearing rodents. In addition, a combination of resveratrol (3 mg/kg body weight) and fish oil was also unable to induce any changes in skeletal muscle weights. CONCLUSIONS: It is therefore concluded from this study that resveratrol is unable to influence muscle mass in vivo and has no potential role as anticachectic agent for the treatment of muscle wasting associated with tumour growth.

Effects of interleukin-15 on lipid oxidation: disposal of an oral [(14)C]-triolein load.

Interleukin 15 (IL-15) has previously been shown to have important effects on lipid metabolism in adipose tissue, particularly influencing the rate of the de novo fatty acid synthesis. The results presented here show that chronic administration to rats (100 microg/kg body weight) has important effects on the metabolic fate of an exogenous [(14)C]-triolein load, decreasing the incorporation of lipid into adipose tissue and significantly increasing the total (14)CO(2) formation from [(14)C]-triolein. Skeletal muscle and possibly liver seem to be the main organs involved in the action of IL-15 on lipid oxidation, since the presence of the cytokine in incubated EDL muscle with [(14)C]-palmitic acid increased (14)CO(2) formation by 39%. Concerning the mechanism, the results suggest that the transport of fatty acids into mitochondria could be involved in the action of IL-15 since the cytokine clearly increases the presence of L-CPT-I and CPT-II in liver tissue. In addition, IL-15 treatment resulted in a significant increment in the gene expression of PPARdelta, a transcription factor clearly related with lipid catabolism in many tissues. Altogether, the results presented here suggest that IL-15 alters exogenous lipid partitioning, limiting adipose tissue uptake and favouring oxidation.

Cancer cachexia results in an increase in TNF-alpha receptor gene expression in both skeletal muscle and adipose tissue.

The mRNA content of both TNF-alpha and its receptors (TNFR1 and TNFR2) in skeletal muscle and adipose tissue from cachectic rats has been assessed. The implantation of the Yoshida AH-130 ascites hepatoma resulted in substantial decrease in skeletal muscle TNF-alpha expression as early as at day 2 following tumour implantation. However, the mRNA content for the two receptors followed a different pattern, being significantly increased at day 7. In adipose tissue the expression of the TNF-alpha gene was significantly increased at all the time points studied, whereas TNF-alpha receptors expression followed a similar pattern to that observed in skeletal muscle. Western blot analysis indicated that the TNFR1 protein followed an identical pattern to that observed in the mRNA content both in muscle and adipose tissue. It is concluded that, during experimental cancer cachexia, the contribution of muscle-produced TNF-alpha is decreased; however, significant changes were observed in relation with TNF-alpha receptors at the skeletal muscle level that could possibly be related to the muscle wasting process associated with tumour growth.

Interleukin-15 decreases proteolysis in skeletal muscle: a direct effect.

Incubation of rat isolated skeletal muscles (extensor digitorum longus) in the presence of 100 ng/ml of human recombinant interleukin-15 (IL-15) resulted in a significant decrease in total proteolytic rate, while it had no effect on total protein synthesis as measured by the incorporation of (14)C-phenylalanine into muscle protein. In addition, IL-15 had no effect on either amino acid uptake (as determined by the tissue uptake of labelled [1-(14)C]MeAIB) or alanine utilization by incubated skeletal muscles. Similarly, a single injection of IL-15 (100 microg/kg) in vivo did not result in any changes in amino acid uptake (as measured by the tissue uptake of alpha-[1-(14)C]AIB) or alanine metabolism, with the exception of alanine carbon incorporation into lipids, which was significantly increased in adipose tissue as a result of IL-15 administration. The results suggest that the main mechanism involved in the anabolic effects of IL-15 in skeletal muscle relies on a decrease in the proteolytic rate.

Interleukin-15 decreases lipid intestinal absorption.

Administration of a single acute intravenous injection of interleukin-15 (IL-15) (100 microg/kg bw) to rats resulted in a significant decrease (22%) in triacylglycerol absorption, as measured by using [14C]-triolein load. The cytokine, however, did not influence the oxidation of the exogenously administered lipid or the tissue uptake of [14C]-triolein; this is in concordance with the lack of effects found in the measurement of the tissue lipoprotein lipase activity. Concerning the mechanism involved in the decreased intestinal absorption associated with IL-15 administration, the results presented clearly demonstrate that changes in gastric emptying and intestinal mobility are not involved, as the effect is specific for triacylglycerols. In conclusion, intestinal absorption may be an additional mechanism to take into consideration to explain the 'anti-fat' effect of this cytokine.

Plasma from pregnant rats has anti-tumoural activity.

Plasma from 12-day pregnant rats showed a marked anti-tumoural activity when added to short-term cultures of rat Yoshida AH-130 cells. Indeed, 24 h after the addition of 10% pregnant rat plasma resulted in a 34% decrease in cell number as compared with cells without any serum additions or those that were added with virgin plasma. The decrease in cell number was associated with the presence of a marked aneuploid peak, as indicated by flow cytometry analysis, which suggests that pregnant plasma induces apoptosis in this particular tumour cell line. Interestingly, addition of human chorionic gonatrophin to the cell cultures did not exert any effects either on cell content of cell cycle distribution, therefore suggesting that the presence of high concentrations of this hormone in pregnant plasma is unlikely to be the cause of the increased cell death.

Effect of c-ski overexpression on the development of cachexia in mice bearing the Lewis lung carcinoma.

Overexpression of the proto-oncogene c-ski in mice results in the development of a hypertrophic phenotype, characterized by increases in body and muscle weights. It has been previously shown in our laboratories that down-regulation of muscle protein breakdown associated with reduced expression of genes pertaining to different proteolytic systems likely account for this hypertrophic pattern. The aim of the present study was to evaluate the resistance of c-ski transgenic mice to catabolic stimuli such as those induced by the growth of the Lewis lung carcinoma. The tumor elicited a loss of body weight either in transgenic or in non-transgenic animals, although it was less pronounced in the former. The mass of gastrocnemius, tibialis and extensor digitorum longus (EDL) muscles were significantly reduced in non-transgenic tumor-bearing mice. Despite the anabolic setting displayed by the transgenic animals, the EDL only is completely protected against wasting. Indeed, gastrocnemius, tibialis and soleus show a reduction in weight, the latter two being significantly more depleted when compared to the non-transgenic tumor bearers. Similarly, the perigenital white adipose tissue presented a reduced mass which was more marked in the transgenic group. The quantitation of gene expression for ubiquitin, E2, C8 and calpain in the EDL showed marked differences between the transgenic and the non-transgenic groups of tumor hosts. As expected from previous results, in the latter group most of the transcripts examined increased with respect to controls as a consequence of tumor growth; by contrast, in the transgenic tumor hosts there was a significant reduction of ubiquitin, E2, C8 subunit, and calpain mRNA levels in comparison with the transgenic tumor-free animals. These results show that c-ski hyperexpression prevents tumor-induced muscle wasting in the EDL muscle, likely by impairing the state of activation of different proteolytic systems. However, the lack of effectiveness in the other muscles examined suggests that the achievement of a significant interference with the development of cachexia at the molecular level is not an easy task and probably should be designed taking into consideration more than one target.

Interleukin-15 is able to suppress the increased DNA fragmentation associated with muscle wasting in tumour-bearing rats.

Administration of interleukin-15 (IL-15) to rats bearing the Yoshida AH-130 ascites hepatoma (a tumour that induces an important cachectic response) resulted in a significant reduction of muscle wasting, both measured as muscle weight and as protein content of different types of skeletal muscle. In addition, the administration of the cytokine completely reversed the increased DNA fragmentation observed in skeletal muscle of tumour-bearing animals. Concerning the mechanism(s) involved in the anti-apoptotic effects of IL-15 on skeletal muscle, the administration of the cytokine resulted in a considerable decrease in both R1 (43%) and R2 (64%) TNF-alpha receptors (TNFRs), and therefore it may be suggested that IL-15 decreases apoptosis by affecting TNF-alpha signalling. Formation of NO could be the signalling event associated with the activation of apoptosis in muscle of tumour-bearing rats; indeed, administration of IL-15 decreased the inducible nitric oxide synthase protein levels by 73%, suggesting that NO formation and muscle apoptosis during tumour growth are related. In conclusion, IL-15 seems to be able to reduce/suppress protein loss and apoptosis related to muscle wasting during cancer cachexia in experimental animals.

Rat liver lipogenesis is modulated by interleukin-15.

Interleukin-15 (IL-15) administration to rats resulted in an important decrease in carcass fat (27%) and, consequently, in white adipose tissue mass. This decrease was linked with a decreased lipogenic rate (per g of tissue) in both adipose tissue (53%) and liver (36%). The decrease in hepatic lipogenesis was associated with lower hepatic citrate levels (49%) and a reduced activity of lipogenic enzymes. The results presented here further demonstrate the involvement of IL-15 in lipid metabolism. Thus, IL-15 seems to be able to modulate de novo fatty acid synthesis, possibly by influencing citrate tissue levels, an allosteric activator of the key lipogenic enzyme acetyl-CoA carboxylase.