Combined targeting of EGFR and HER2 against prostate cancer stem cells.

Progression of prostate cancer has been associated with EGFR and HER2 activation and to tumor-initiating cells contribution toward chemotherapy resistance. We investigated the efficacy of a dual intervention against EGFR and HER2 to deplete the tumor-initiating cells, optimize the chemotherapy management and prevent the progression of castration-resistant prostate cancer (CRPC) cells. Using DU145, PC3, and 22Rv1 CRPC cell lines, biochemical analysis revealed activation of EGFR, HER2, MAPK, and STAT3 in DU145 and 22Rv1, and AKT and SRC in DU145 and PC-3. pSTAT3 nuclear staining was observed in DU145 xenografts and in 12 out of 14 CRPC specimens. The in vivo dual targeting of ErbB receptors with Cetuximab and Trastuzumab combined with chemotherapy caused an effective antitumor response in DU145 xenografted mice displaying STAT3 activation; conversely PC-3 bearing mice experienced tumor relapse. The potentiating of in vivo cytotoxic effect in DU145 model was accompanied by a significant decrease of prostatosphere-forming capacity assessed in vitro on residual tumor cells. Additionally, combined treatment in vitro with Cetuximab, Trastuzumab and chemotherapy negatively affected DU145 and 22Rv1 sphere formation, suggesting the critical function of ErbB receptors for tumor-initiating cells proliferation; no effect on PC-3 clonogenic potential was observed, indicating that other receptors than EGFR and HER2 may sustain PC3 tumor-initiating cells. These findings provided the preclinical evidence that the dual inhibition of EGFR and HER2 by targeting tumor-initiating cells may improve the efficacy of the current chemotherapy regimen, bringing benefits especially to castration-resistant patients with activated STAT3, and preventing disease progression.

c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.

BACKGROUND: Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. METHODS: The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. RESULTS AND CONCLUSIONS: We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. GENERAL SIGNIFICANCE: Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis.

miR-9 and miR-200 Regulate PDGFRß-Mediated Endothelial Differentiation of Tumor Cells in Triple-Negative Breast Cancer.

Organization of cancer cells into endothelial-like cell-lined structures to support neovascularization and to fuel solid tumors is a hallmark of progression and poor outcome. In triple-negative breast cancer (TNBC), PDGFRß has been identified as a key player of this process and is considered a promising target for breast cancer therapy. Thus, we aimed at investigating the role of miRNAs as a therapeutic approach to inhibit PDGFRß-mediated vasculogenic properties of TNBC, focusing on miR-9 and miR-200. In MDA-MB-231 and MDA-MB-157 TNBC cell lines, miR-9 and miR-200 promoted and inhibited, respectively, the formation of vascular-like structures in vitro Induction of endogenous miR-9 expression, upon ligand-dependent stimulation of PDGFRß signaling, promoted significant vascular sprouting of TNBC cells, in part, by direct repression of STARD13. Conversely, ectopic expression of miR-200 inhibited this sprouting by indirectly reducing the protein levels of PDGFRß through the direct suppression of ZEB1. Notably, in vivo miR-9 inhibition or miR-200c restoration, through either the generation of MDA-MB-231-stable clones or peritumoral delivery in MDA-MB-231 xenografted mice, strongly decreased the number of vascular lacunae. Finally, IHC and immunofluorescence analyses in TNBC specimens indicated that PDGFRß expression marked tumor cells engaged in vascular lacunae. In conclusion, our results demonstrate that miR-9 and miR-200 play opposite roles in the regulation of the vasculogenic ability of TNBC, acting as facilitator and suppressor of PDGFRß, respectively. Moreover, our data support the possibility to therapeutically exploit miR-9 and miR-200 to inhibit the process of vascular lacunae formation in TNBC. Cancer Res; 76(18); 5562-72. ©2016 AACR.

Influence of fatty acid-free diet on mammary tumor development and growth rate in HER-2/Neu transgenic mice.

Numerous investigations have found a relationship between higher risk of cancer and increased intake of fats, while results of clinical studies of fat reduction and breast cancer recurrence have been mixed. A diet completely free of fats cannot be easily administered to humans, but experimental studies in mice can be done to determine whether this extreme condition influences tumor development. Here, we examined the effects of a FA-free diet on mammary tumor development and growth rate in female FVB-neu proto-oncogene transgenic mice that develop spontaneous multifocal mammary tumors after a long latency period. Mice were fed a fatty acid-free diet beginning at 112, 35, and 30 days of age. In all these experiments, tumor appearance was delayed, tumor incidence was reduced and the mean number of palpable mammary tumors per mouse was lower, as compared to standard diet-fed mice. By contrast, tumor growth rate was unaffected in mice fed the fatty acid-free diet. Plasma of mice fed the fatty acid-free diet revealed significantly higher contents of oleic, palmitoleic and 20:3ω9 acids and lower contents of linoleic and palmitic acids. In conclusion, these findings indicate that a FA-free diet reduces tumor incidence and latency but not tumor growth rate, suggesting that a reduction in dietary FAs in humans may have a protective effect on tumorigenesis but not on tumors once they appear.

EGFR through STAT3 modulates ΔN63α expression to sustain tumor-initiating cell proliferation in squamous cell carcinomas.

Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR-ΔNp63α axis in proliferation of SCC tumor-initiating cells (TICs). SCC cell lines A-431, Cal-27, and SCC-25 treated with EGF showed a time-dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non-differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non-differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR-ΔNp63α axis is crucial for proliferation of TICs present in SCCs.

Surveillance of spontaneous breast cancer metastasis by TRAIL-expressing CD34⁺ cells in a xenograft model.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), delivered as a membrane-bound molecule expressed on the surface of adenovirus-transduced CD34(+) cells (CD34-TRAIL(+)), was analyzed for its apoptotic activity in vitro on 12 breast cancer cell lines representing estrogen receptor-positive, HER2(+) and triple-negative (TN) subtypes and for its effect on tumor growth, vascularization, necrosis, and lung metastasis incidence in NOD/SCID mice xenografted with the TN breast cancer line MDA-MB-231. Mesenchymal TN cell lines, which are the richest in putative tumor stem cells among the different breast cancer cell subtypes, were the most susceptible to apoptosis induced by CD34-TRAIL(+) cells. Indeed, tumor cell "stemness", assessed based on the proportion of CD44(+)/CD24(-/low) cells, was significantly correlated with susceptibility to TRAIL. Moreover, in vitro cytotoxicity experiments showed that CD34-TRAIL(+) cells selectively targeted CD44(+)/CD24(-/low) cells. Although in vivo treatment with CD34-TRAIL(+) cells did not lead to tumor growth inhibition, treated mice revealed significantly larger areas of necrosis associated with damage of tumor vasculature than did control mice. Moreover, lungs from MDA-MD-231 tumor-bearing mice were completely free of metastases at 12 days after the last injection of CD34-TRAIL(+) cells, whereas metastases were present in all control mouse lungs. An anti-metastatic effect of CD34-TRAIL(+) cells was also observed in a model of experimental lung metastases. The correlation between in vitro susceptibility to membrane-bound TRAIL and tumor stem cell content, together with CD34-TRAIL(+) cell-induced inhibition of the metastatic process, points to the selective targeting of cancer stem cells by CD34-armed cells and the potential value of such cells in eradicating tumor stem cells before the onset of overt metastases.

The human splice variant Δ16HER2 induces rapid tumor onset in a reporter transgenic mouse.

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform "per se" mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein.

Influence of lignans depletion on murine mammary gland morphology.

Different studies have focused on the effects of phytoestrogens-supplemented diets on mammary gland morphogenesis and breast cancer risk; however, particular dieting behaviors and food choices may result in a reduction of the natural source of phytoestrogens. The evaluation of a reduced phytoestrogens intake effect by depletion without modifying other dietary ingredients is hard. Since lignans, the largest contributors to phytoestrogens intake in Western diets, are metabolized into bioactive compounds by gut bacteria, long-term antibiotic treatments, inducing intestinal microflora disruption, may reduce enterolactone availability. To elucidate the effect of phytoestrogens lack on mammary tissue morphogenesis, female FVB mice were treated with gentamicin or metronidazole/ciprofloxacin from the age of 6 to 7 wk. After 21 wk, enterolactone urine levels were 120.07 +/- 20.5 ng/ml in untreated mice, 30.4 +/- 24.46 ng/ml in metronidazole/ciprofloxacin-treated mice, and 3.29 +/- 4.38 ng/ml in gentamicin-treated mice. Histological analysis revealed no significant alterations of mammary morphology in metronidazole/ciprofloxacin-treated mice, whereas gentamicin-treated mice showed increase of ducts number and duct-tree branching vs. controls. These findings indicate that normal mammary tissue size and shape are maintained even in the presence of low levels of lignans and suggest that only a complete depletion of these compounds induced significant alterations of mammary gland structure.

The total activity of a mixture of okadaic acid-group compounds can be calculated by those of individual analogues in a phosphoprotein phosphatase 2A assay.

Monitoring of okadaic acid (OA)-group toxins in seafood is of paramount importance for the protection of consumer health from diarrheic shellfish poisoning. The property of OA-group compounds to inhibit type 2A serine/threonine phosphoprotein phosphatase (PP2A) has been exploited for the detection of OA in several experimental settings, but the performance of PP2A inhibition assays in the quantification of mixtures of OA-group compounds has not been reported yet. We have used a PP2A inhibition assay to analyze the total effect of mixtures including OA and one of its analogues, okadaol (OOH), by measuring the activity of individual compounds and of toxin mixtures through the inhibition they exert on the PP2A enzyme. We found that both OA and OOH inhibit PP2A under our experimental conditions, with IC50 values of 0.37 +/- 0.04 nM and 4.3 +/- 0.8 nM, respectively, confirming that OOH is about ten-fold less potent than OA. PP2A assays were also carried out with predefined mixtures of OA and OOH, covering the full dose-response of one compound in the presence of increasing concentrations of the other toxin. The experimental data we obtained were used to analyze their correlation with those that could be calculated by adding the relative effects exerted by individual analogues, and we found that a good correlation exists between the observed and the expected data, when the predicted effect was calculated on the basis of toxicity equivalence factors. Our findings show that an additive model based on the use of toxicity equivalence factors of individual toxins is appropriate for the calculation of the total activity of multi-component mixtures of OA-group compounds in unknown samples.

Critical role of TLR9 in acute graft-versus-host disease.

Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9(-/-)) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4(-/-)) mice. The reduced mortality and morbidity in TLR9(-/-) mice related with reduced stimulatory activity of TLR9(-/-) spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9(+/+) into TLR9(-/-) and TLR9(-/-) into TLR9(+/+) chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9(+/+) cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation.

Sodium-dependent glucose transporter-1 as a novel immunological player in the intestinal mucosa.

In this study, we demonstrate the protective effect of the activation of sodium-dependent glucose transporter-1 (SGLT-1) on damages induced by TLR ligands, in intestinal epithelial cells and in a murine model of septic shock. In intestinal epithelial cell lines, glucose inhibited the IL-8/keratinocyte-derived chemokine production and the activation of the TLR-related transcription factor NF-kappaB stimulated by LPS or CpG-oligodeoxynucleotide. Oral ingestion of glucose was found to protect 100% of mice from lethal endotoxic shock induced by i.p. LPS administration; protection was only observed when glucose was administered orally, not by i.p. route, suggesting the important role of intestinal epithelial cells in this protection. In addition, we observed that the in vivo protection depends on an increase of anti-inflammatory cytokine IL-10. The cornerstone of the observed immunomodulatory and life-saving effects resides in activation of SGLT-1; in fact, the glucose analog 3-O-methyl-d-gluco-pyranose, which induces the transporter activity, but is not metabolized, exerted the same inhibitory effects as glucose both in vitro and in vivo. Thus, we propose that activated SGLT-1, apart from its classical metabolic function, may be a promising target for inhibition of bacteria-induced inflammatory processes and life-saving treatments, assuming a novel role as an immunological player.

Activation of enteroendocrine cells via TLRs induces hormone, chemokine, and defensin secretion.

Enteroendocrine cells are known primarily for their production of hormones that affect digestion, but they might also be implicated in sensing and neutralizing or expelling pathogens. We evaluate the expression of TLRs and the response to specific agonists in terms of cytokines, defensins, and hormones in enteroendocrine cells. The mouse enteroendocrine cell line STC-1 and C57BL/6 mice are used for in vitro and in vivo studies, respectively. The presence of TLR4, 5, and 9 is investigated by RT-PCR, Western blot, and immunofluorescence analyses. Activation of these receptors is studied evaluating keratinocyte-derived chemokine, defensins, and cholecystokinin production in response to their specific agonists. In this study, we show that the intestinal enteroendocrine cell line STC-1 expresses TLR4, 5, and 9 and releases cholecystokinin upon stimulation with the respective receptor agonists LPS, flagellin, and CpG-containing oligodeoxynucleotides. Release of keratinocyte-derived chemokine and beta-defensin 2 was also observed after stimulation of STC-1 cells with the three TLR agonists, but not with fatty acids. Consistent with these in vitro data, mice showed increased serum cholecystokinin levels after oral challenge with LPS, flagellin, or CpG oligodeoxynucleotides. In addition to their response to food stimuli, enteroendocrine cells sense the presence of bacterial Ags through TLRs and are involved in neutralizing intestinal bacteria by releasing chemokines and defensins, and maybe in removing them by releasing hormones such as cholecystokinin, which induces contraction of the muscular tunica, favoring the emptying of the distal small intestine.

Influence of antibiotic treatment on breast carcinoma development in proto-neu transgenic mice.

The effect of prolonged antibiotic treatments on tumor development was evaluated in proto-neu transgenic mice, which spontaneously develop mammary carcinomas. Virgin transgenic mice were treated with metronidazole/ciprofloxacin or gentamicin through the drinking water. The hazard ratio [HR; 95% confidence interval (95% CI)] of breast cancer occurrence in metronidazole/ciprofloxacin-treated mice was more than triple that for controls [3.11 (1.13-8.53); P = 0.028], whereas only a slight increase in HR (95% CI) was observed in gentamicin-treated mice [1.39 (0.56-3.47); P = 0.481]. Tumor growth rate in gentamicin-treated mice was significantly faster than in untreated control mice (P = 0.043). Moreover, mammary glands from mice treated with either antibiotic regimen showed increased lobulization, with more numerous and more developed terminal ductal lobular units than in controls. These results indicate that prolonged exposure to relevant doses of antibiotics affects the mammary glands in this particular model of HER-2/neu transgenic mice; further studies to understand the precise mechanism by which antibiotic treatments influence mammary gland differentiation are critical.

Antitumor activity of the TLR-5 ligand flagellin in mouse models of cancer.

Flagellin, the structural protein subunit of the bacterial flagellum, is specifically recognized by TLR-5 and has potent immunomodulatory effects. The antitumor effects of purified Salmonella typhimurium flagellin were evaluated in mice transplanted s.c. with a weakly immunogenic murine tumor or with its variant stably transfected to express the highly antigenic human HER-2 oncoprotein. Peritumoral administration of flagellin 8-10 days after tumor implantation did not affect the growth rate of the weakly immunogenic tumor but significantly inhibited growth of the antigenic variant tumor. In contrast, flagellin administered at the time of implantation of the antigenic tumor led to accelerated tumor growth. These contrasting effects of flagellin on tumor growth correlated with the type of immune response induced; i.e., late flagellin administration was associated with an increased IFN-gamma:IL-4 ratio and the decreased frequency of CD4+CD25+ T regulatory cells, whereas flagellin treatment at the time of tumor implantation decreased the IFN-gamma:IL-4 ratio and increased CD4+CD25+ T cell frequency. When the early flagellin treatment was combined with administration of CpG-containing oligodeoxynucleotides, tumor growth was completely suppressed, indicating synergy between flagellin and CpG-containing oligodeoxynucleotides. Together, these data provide evidence that flagellin can have contrasting effects on tumor growth.

Therapeutic synergism of gemcitabine and CpG-oligodeoxynucleotides in an orthotopic human pancreatic carcinoma xenograft.

CpG-oligodeoxynucleotides (CpG-ODN) exhibit potent immunostimulatory activity by binding with Toll-like receptor 9 (TLR9). Based on the finding that TLR9 is highly expressed and functional in pancreatic tissue, we evaluated the antitumor effects of chemotherapy combined with CpG-ODNs in the orthotopic mouse model of a human pancreatic tumor xenograft. Chemotherapy consisted of the maximum tolerated dose of gemcitabine (i.v., 100 mg/kg, q3dx4). CpG-ODNs were delivered (i.p., 20 microg/mouse), weekly, after the end of chemotherapy. CpG-ODNs alone had little effect on tumor growth, whereas gemcitabine alone significantly delayed the median time of disease onset (palpable i.p. tumor) and of bulky disease development (extensive peritoneal tumor burden), but did not enhance survival time. When the gemcitabine regimen was followed by administration of the immunostimulator, development of bulky disease was delayed, survival time was significantly improved (median survival time, 106 days; P < 0.02 versus gemcitabine-treated mice). Autoptic examination showed that tumor spread in the peritoneal cavity was reduced to a greater extent than with gemcitabine alone. All treatment regimens were well-tolerated. The use of nude mice excluded a T cell-mediated immune response, whereas the high pancreatic expression of TLR9 might have contributed to the tumor response. The clear improvement of survival observed in an orthotopic murine model of human pancreatic cancer by the combined use of CpG-ODNs with chemotherapy suggests the promise of this therapeutic regimen in the clinical setting.

CpG-Oligodeoxynucleotides activate tyrosinase-related protein 2-specific T lymphocytes but do not lead to a protective tumor-specific memory response.

PURPOSE: Peritumoral CpG-oligodeoxynucleotide (ODN) treatment has been successful in tumor mouse models expressing strong antigens to induce activation of tumor-specific CD8+ T lymphocytes which contribute to the control of tumor growth. To get near to clinical reality, the tumor-specific CD8+ response was investigated in mice bearing the weakly immunogenic B16 melanoma tumor and using the melanocyte differentiation tyrosinase-related protein 2 (TRP-2) as a tracking antigen. METHODS: The expansion and activation of TRP-2-specific T lymphocytes by CpG-ODNs was analyzed by tetramer staining and IFN-gamma production assays, while the activity of these cells in both memory and primary response was evaluated in vivo. RESULTS: After CpG-ODN treatment, the number of TRP-2 tetramer-stained CD8+ T lymphocytes was not significantly modified, but these cells produced higher levels of interferon gamma (IFN-gamma) in response to the antigen than those from untreated mice. Mice possessing these activated T lymphocytes, when evaluated for their antitumor memory response, showed marginal protection against intravenous (i.v.) and subcutaneous (s.c.) tumor rechallenge. These cells were not crucial for the control of primary tumor growth since strong reduction of subcutaneous tumor was observed after CpG-ODN treatment in both CD8+ T cell depleted or nondepleted mice. On the contrary, NK cell depletion markedly reduced CpG-ODN-induced tumor growth inhibition. CONCLUSIONS: Altogether, these data indicate the CpG treatment activates tumor-reactive effector CD8+ T lymphocytes, but, paralleling recent clinical observations, our model indicates that the mere activation of antitumor T cells is insufficient to result in a clinical response.