Targeted cancer therapy--are the days of systemic chemotherapy numbered?

Targeted therapy or molecular targeted therapy has been defined as a type of treatment that blocks the growth of cancer cells by interfering with specific cell molecules required for carcinogenesis and tumor growth, rather than by simply interfering with all rapidly dividing cells as with traditional chemotherapy. There is a growing number of FDA approved monoclonal antibodies and small molecules targeting specific types of cancer suggestive of the growing relevance of this therapeutic approach. Targeted cancer therapies, also referred to as "Personalized Medicine", are being studied for use alone, in combination with other targeted therapies, and in combination with chemotherapy. The objective of personalized medicine is the identification of patients that would benefit from a specific treatment based on the expression of molecular markers. Examples of this approach include bevacizumab and olaparib, which have been designated as promising targeted therapies for ovarian cancer. Combinations of trastuzumab with pertuzumab, or T-DM1 and mTOR inhibitors added to an aromatase inhibitor are new therapeutic strategies for breast cancer. Although this approach has been seen as a major step in the expansion of personalized medicine, it has substantial limitations including its high cost and the presence of serious adverse effects. The Cancer Genome Atlas is a useful resource to identify novel and more effective targets, which may help to overcome the present limitations. In this review we will discuss the clinical outcome of some of these new therapies with a focus on ovarian and breast cancer. We will also discuss novel concepts in targeted therapy, the target of cancer stem cells.

Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFĸB pathway.

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.

Stem-like ovarian cancer cells can serve as tumor vascular progenitors.

Neovascularization is required for solid tumor maintenance, progression, and metastasis. The most described contribution of cancer cells in tumor neovascularization is the secretion of factors, which attract various cell types to establish a microenvironment that promotes blood vessel formation. The cancer stem cell hypothesis suggests that tumors are composed of cells that may share the differentiation capacity of normal stem cells. Similar to normal stem cells, cancer stem cells (CSCs) have the capacity to acquire different phenotypes. Thus, it is possible that CSCs have a bigger role in the process of tumor neovascularization. In this study, we show the capacity of a specific population of ovarian cancer cells with stem-like properties to give rise to xenograft tumors containing blood vessels, which are lined by human CD34+ cells. In addition, when cultured in high-density Matrigel, these cells mimic the behavior of normal endothelial cells and can form vessel-like structures in 24 hours. Microscopic analysis showed extensive branching and maturation of vessel-like structures in 7 days. Western blot and flow cytometry analysis showed that this process is accompanied by the acquisition of classic endothelial markers, CD34 and VE-cadherin. More importantly, we show that this process is vascular endothelial growth factor-independent, but IKK beta-dependent. Our findings suggest that anti-angiogenic therapies should take into consideration the inherent capacity of these cells to serve as vascular progenitors.

NV-128, a novel isoflavone derivative, induces caspase-independent cell death through the Akt/mammalian target of rapamycin pathway.

BACKGROUND: Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase-independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV-128 as an inducer of cell death through a caspase-independent pathway. METHODS: Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV-128, and the concentration that caused 50% growth inhibition (GI(50)) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein-protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model. RESULTS: NV-128 was able to induce significant cell death in both paclitaxel-resistant and carboplatin-resistant EOC cells with a GI(50) between 1 microg/mL and 5 microg/mL. Cell death was characterized by chromatin condensation but was caspase-independent. The activated pathway involved the down-regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin-1 followed by nuclear translocation of endonuclease G. CONCLUSIONS: The authors characterized a novel compound, NV-128, which inhibits mTOR and promotes caspase-independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase-dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV-128, which may have beneficial effects in patients with chemoresistant ovarian cancer.

Development and validation of a protein-based signature for the detection of ovarian cancer.

To overcome the significant mortality associated with ovarian cancer, a highly sensitive and specific screening test is urgently needed. CA-125 testing is used to monitor response to chemotherapy, detect recurrence, and detect late stage ovarian cancer. However, CA-125 testing, alone or in combination with ultrasonography, has not been adequate for early detection of ovarian cancer. This article discusses the authors' recent report of a novel multiplex assay that uses a panel of six serum biomarkers: leptin, prolactin, osteopontin, insulin-like growth factor II (IGF-II), macrophage inhibitory factor (MIF), and CA-125. The combination of these six proteins yielded 95.3% sensitivity and 99.4% specificity. The application of this test in the clinical context and the most appropriate population, which could benefit from the test, are discussed.

Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance.

A major burden in the treatment of ovarian cancer is the high percentage of recurrence and chemoresistance. Cancer stem cells (CSCs) provide a reservoir of cells that can self-renew, can maintain the tumor by generating differentiated cells [non-stem cells (non-CSCs)] which make up the bulk of the tumor and may be the primary source of recurrence. We describe the characterization of human ovarian cancer stem cells (OCSCs). These cells have a distinctive genetic profile that confers them with the capacity to recapitulate the original tumor, proliferate with chemotherapy, and promote recurrence. CSC identified in EOC cells isolated form ascites and solid tumors are characterized by: CD44+, MyD88+, constitutive NFkappaB activity and cytokine and chemokine production, high capacity for repair, chemoresistance to conventional chemotherapies, resistance to TNFalpha-mediated apoptosis, capacity to form spheroids in suspension, and the ability to recapitulate in vivo the original tumor. Chemotherapy eliminates the bulk of the tumor but it leaves a core of cancer cells with high capacity for repair and renewal. The molecular properties identified in these cells may explain some of the unique characteristics of CSCs that control self-renewal and drive metastasis. The identification and cloning of human OCSCs can aid in the development of better therapeutic approaches for ovarian cancer patients.

TLR6 modulates first trimester trophoblast responses to peptidoglycan.

Intrauterine bacterial infections are a well-established cause of pregnancy complications. One key observation in a number of abnormal pregnancies is that placental apoptosis is significantly elevated. First trimester trophoblast cells are known to express TLR1 and TLR2 and to undergo apoptosis following exposure to Gram-positive bacterial peptidoglycan (PDG). Thus, the objectives of this study were to determine whether PDG-induced pregnancy complications are associated with placental apoptosis and to characterize the cellular mechanisms involved. We have demonstrated, using an animal model, that delivery of PDG to pregnant mice early in gestation resulted in highly elevated placental apoptosis, evidenced by trophoblast M-30 and active caspase 3 immunostaining. Using an in vitro model of human first trimester trophoblasts, apoptosis induced by PDG was found to be mediated by both TLR1 and TLR2 and that this could be blocked by the presence of TLR6. Furthermore, in the presence of TLR6, exposure to PDG resulted in trophoblast NF-kappaB activation and triggered these cells to secrete IL-8 and IL-6. The findings of this study suggest that a Gram-positive bacterial infection, through TLR2 and TLR1, may directly promote the elevated trophoblast cell death and that this may be the underlying mechanism of pregnancy complications, such as preterm delivery. Furthermore, the expression of TLR6 may be a key factor in determining whether the response to PDG would be apoptosis or inflammation.

Diagnostic markers for early detection of ovarian cancer.

PURPOSE: Early detection would significantly decrease the mortality rate of ovarian cancer. In this study, we characterize and validate the combination of six serum biomarkers that discriminate between disease-free and ovarian cancer patients with high efficiency. EXPERIMENTAL DESIGN: We analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All six markers were evaluated in a training set (181 samples from the control group and 113 samples from OC patients) and a test set (181 sample control group and 43 ovarian cancer). RESULTS: Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves were good enough to differentiate healthy versus cancer cells. However, the combination of the six markers provided a better differentiation than CA-125. Four models with <2% classification error in training sets all had significant improvement (sensitivity 84%-98% at specificity 95%) over CA-125 (sensitivity 72% at specificity 95%) in the test set. The chosen model correctly classified 221 out of 224 specimens in the test set, with a classification accuracy of 98.7%. CONCLUSIONS: We describe the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. This novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer.

A novel three-dimensional in vitro system to study trophoblast-endothelium cell interactions.

INTRODUCTION: Pregnancy complications have been linked to improper trophoblast migration and failure of spiral artery transformation. Endothelial cells play an essential role in directing trophoblast migration and transformation, although by an unknown mechanism. We describe a novel in vitro model to evaluate endothelial-trophoblast interaction and signaling in a three-dimensional system. METHOD OF STUDY: Immortalized human endometrial endothelial cell line and first trimester trophoblast cells were co-cultured. Endothelial transformation into vessel-like structures occurred in Matrigel(TM) OpenLab Image Analysis software was used to monitor labeled trophoblast migration and endothelium transformation. Cytokine/chemokine production was determined using Multiplex. RESULTS: Trophoblast migrates toward endothelial cells in Matrigel, aligns on top of the endothelium within 4-8 hr and achieves complete replacement of the endothelium by 72-96 hr. Lipopolysaccharide treatment damages the endothelium and disrupts endothelium-trophoblast interaction. CONCLUSION: We report a novel three-dimensional in vitro and in vivo system of trophoblast-endothelium cell interaction. Significant changes in endothelial cells' phenotype are observed upon differentiation in Matrigel. These changes may be necessary for endothelium to direct trophoblast migration and transformation.

Macrophage migration inhibitory factor expression in ovarian cancer.

OBJECTIVE: We evaluated the hypothesis that ovarian cancer patients have significantly higher levels of serum macrophage migration inhibitory factor (MIF). STUDY DESIGN: MIF levels were determined by enzyme-linked immunosorbent assay (ELISA) in epithelial ovarian cancer cell lines and immortalized normal ovarian surface epithelial cells and in serum of ovarian cancer patients (n = 54) and age-matched healthy women (n = 60). To determine the impact of Toll-like receptor-4 ligation on MIF levels, cells were treated for 48 hours with lipopolysaccharide. RESULTS: Cancer cells, but not normal cells, secrete significant amounts of MIF. This correlates in vivo, where serum MIF levels are significantly higher in ovarian cancer patients. Treatment of cancer cells with lipopolysaccharide induced a significant increase in MIF secretion. CONCLUSION: MIF may be relevant in the process of ovarian cancer formation and progression. The events leading to the induction of MIF expression and its contribution to ovarian cancer progression may open new venues for targeted therapy.

XAF1 mediates tumor necrosis factor-alpha-induced apoptosis and X-linked inhibitor of apoptosis cleavage by acting through the mitochondrial pathway.

Tumor necrosis factor-alpha (TNF-alpha) and Fas ligand induce apoptosis by interacting with their corresponding membrane-bound death receptors and activating caspases. Since both systems share several components of the intracellular apoptotic cascade and are expressed by first trimester trophoblasts, it is unknown how these cells remain resistant to Fas ligand while sensitive to TNF-alpha. XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a proapoptotic protein that antagonizes the caspase-inhibitory activity of XIAP. Here, we demonstrated that XAF1 functions as an alternative pathway for TNF-alpha-induced apoptosis by translocating to the mitochondria and promoting XIAP inactivation. In addition, we showed that the overexpression of XAF1 sensitized first trimester trophoblast cells to Fas-mediated apoptosis. Furthermore, we also determined that the differential expression of XAF1 in first and third trimester trophoblast cells was due to changes in XAF1 gene methylation. Our results establish a novel regulatory pathway controlling trophoblast cell survival and provide a molecular mechanism to explain trophoblast sensitivity to TNF-alpha and the increased number of apoptotic trophoblast cells observed near term. Aberrant XAF1 expression and/or localization may have consequences for normal pregnancy outcome.

Expression and secretion of antiviral factors by trophoblast cells following stimulation by the TLR-3 agonist, Poly(I : C).

BACKGROUND: During pregnancy, the placenta may become exposed to micro-organisms, such as viruses, which may pose a substantial threat to the embryo/fetus well-being. Recent insight into the immunological capabilities of the trophoblast suggests that the placenta may function as an active barrier by recognizing and responding to pathogens through Toll-like receptors (TLRs). METHODS: The objective of this study was to determine whether the engagement of TLR-3 with viral dsRNA by first-trimester trophoblast could induce the production of factors necessary to generate an antiviral response. Therefore, trophoblast cells were exposed to the TLR-3 agonist, Poly(I : C). RESULTS: We report that following stimulation with Poly(I : C), first-trimester trophoblast cells produce interferon beta (IFNbeta) and secretory leukocyte protease inhibitor (SLPI), as well as the intracellular factors 2',5'-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). This response is TLR-3 specific because the TLR-4 ligand, lipopolysaccharide (LPS), had no effect on the production of these antimicrobial factors. Furthermore, we describe a positive feedback mechanism in which IFNbeta enhances the antiviral response by promoting the production of OAS, MxA and APOBEC3G. CONCLUSIONS: These findings suggest that trophoblast cells are able to recognize and specifically respond to viral products in a highly regulated fashion and that the placenta may be pivotal in the control of viral infections at the maternal-fetal interface.

TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer.

Evidence suggests that an inflammatory profile of cytokines and chemokines persisting at a particular site would lead to the development of a chronic disease. Recent studies implicate bacterial infection as one possible link between inflammation and carcinogenesis; however, the crucial molecular pathways involved remain unknown. We hypothesized that one possible upstream signaling pathway leading to inflammation in carcinogenesis may be mediated by Toll-like receptors (TLR). We describe for the first time an adaptive mechanism acquired by ovarian cancer cells that allows them to promote a proinflammatory environment and develop chemoresistance. We propose that the TLR-4-MyD88 signaling pathway may be a risk factor for developing cancer and may represent a novel target for the development of biomodulators. Our work explains how bacterial products, such as lipopolysaccharide, can promote, directly from the tumor, the production of proinflammatory cytokines and the enhancement of tumor survival. In addition, we provide new evidence that links TLR-4 signaling, inflammation, and chemoresistance in ovarian cancer cells.

A role for TLRs in the regulation of immune cell migration by first trimester trophoblast cells.

Normal pregnancy is characterized by the presence of innate immune cells at the maternal-fetal interface. Originally, it was postulated that the presence of these leukocytes was due to an immune response toward paternal Ags expressed by the invading trophoblasts. Instead, we and others postulate that these innate immune cells are necessary for successful implantation and pregnancy. However, elevated leukocyte infiltration may be an underlying cause of pregnancy complications, such as preterm labor or preeclampsia. Furthermore, such conditions have been attributed to an intrauterine infection. Therefore, we hypothesize that first trimester trophoblast cells, upon recognition of microbes through TLRs, may coordinate an immune response by recruiting cells of the innate immune system to the maternal-fetal interface. In this study, we have demonstrated that human first trimester trophoblast cells constitutively secrete the chemokines growth-related oncogene, growth-related oncogene alpha, IL-8, and MCP-1 and are able to recruit monocytes and NK cells, and to a lesser degree, neutrophils. Following the ligation of TLR-3 by the viral ligand, poly(I:C), or TLR-4 by bacterial LPS, trophoblast secretion of chemokines is significantly increased and this in turn results in elevated monocyte and neutrophil chemotaxis. In addition, TLR-3 stimulation also induces trophoblast cells to secrete RANTES. These results suggest a novel mechanism by which first trimester trophoblast cells may differentially modulate the maternal immune system during normal pregnancy and in the presence of an intrauterine infection. Such altered trophoblast cell responses might contribute to the pathogenesis of certain pregnancy complications.

Serum protein markers for early detection of ovarian cancer.

Early diagnosis of epithelial ovarian cancer (EOC) would significantly decrease the morbidity and mortality from this disease but is difficult in the absence of physical symptoms. Here, we report a blood test, based on the simultaneous quantization of four analytes (leptin, prolactin, osteopontin, and insulin-like growth factor-II), that can discriminate between disease-free and EOC patients, including patients diagnosed with stage I and II disease, with high efficiency (95%). Microarray analysis was used initially to determine the levels of 169 proteins in serum from 28 healthy women, 18 women newly diagnosed with EOC, and 40 women with recurrent disease. Evaluation of proteins that showed significant differences in expression between controls and cancer patients by ELISA assays yielded the four analytes. These four proteins then were evaluated in a blind cross-validation study by using an additional 106 healthy females and 100 patients with EOC (24 stage I/II and 76 stage III/IV). Upon sample decoding, the results were analyzed by using three different classification algorithms and a binary code methodology. The four-analyte test was further validated in a blind binary code study by using 40 additional serum samples from normal and EOC cancer patients. No single protein could completely distinguish the cancer group from the healthy controls. However, the combination of the four analytes exhibited the following: sensitivity 95%, positive predictive value (PPV) 95%, specificity 95%, and negative predictive value (NPV) 94%, a considerable improvement on current methodology.

Investigation of the role of B-cells in type 1 diabetes in the NOD mouse.

B-cells are important in the development of type 1 diabetes, but their role is not completely defined. Although B-cells produce autoantibodies, these are not thought to be pathogenic; however, their antigen-presenting function is postulated to be critical. To examine the relative importance of these functions of B-cells, we have generated nonobese diabetic (NOD) B-cell-deficient mice that express a transgene encoding a mutant heavy chain immunoglobulin transgene on the cell surface but cannot secrete immunoglobulins (mIgs). This allowed us to dissect the importance of the relative roles of antigen presentation, dissociated from antibody production. We found that the expression of the mIg transgene increased insulitis and the incidence of diabetes compared with transgene-negative NOD B-cell-deficient mice, indicating that the ability to produce antibodies is not necessary for B-cells to have some effect on the development of diabetes. However, diabetes was not restored to the level seen in normal NOD mice. This may relate to reduced ability to activate an islet-specific T-cell repertoire, presumably due to the reduced islet-specific B-cell repertoire. Our results implicate a specific antigen-presenting function for B-cells.

Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen.

Allergic asthma is an inflammatory lung disease initiated and directed by T helper cells type 2 (Th2). The mechanism involved in generation of Th2 responses to inert inhaled antigens, however, is unknown. Epidemiological evidence suggests that exposure to lipopolysaccharide (LPS) or other microbial products can influence the development and severity of asthma. However, the mechanism by which LPS influences asthma pathogenesis remains undefined. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 (Th1) responses, it is unclear if TLRs are needed for Th2 priming. Here, we report that low level inhaled LPS signaling through TLR4 is necessary to induce Th2 responses to inhaled antigens in a mouse model of allergic sensitization. The mechanism by which LPS signaling results in Th2 sensitization involves the activation of antigen-containing dendritic cells. In contrast to low levels, inhalation of high levels of LPS with antigen results in Th1 responses. These studies suggest that the level of LPS exposure can determine the type of inflammatory response generated and provide a potential mechanistic explanation of epidemiological data on endotoxin exposure and asthma prevalence.

Resident lung antigen-presenting cells have the capacity to promote Th2 T cell differentiation in situ.

Antigen exposure via airway epithelia is often associated with a failure to prime or with the preferential priming of Th2 cells. We previously reported that the intranasal delivery of a Th1-inducing antigen promoted Th2-dominated responses, rather than the expected Th1 responses. Thus, we proposed that when pulmonary T cell priming is induced, the lung microenvironment might intrinsically favor the generation of Th2 types of responses. To establish a potential mechanism for such preferential priming, we examined the initial interactions between antigens and resident antigen-presenting cells (APCs) within the lung. We show that intranasally delivered antigens are preferentially taken up and can be presented to antigen-specific T cells by a resident population of CD11c(bright) APCs. Most of these antigen-loaded APCs remained within lung tissues, and migration into secondary lymphoid organs was not crucial for T cell priming to occur within the pulmonary tract. Furthermore, these pulmonary APCs demonstrated a marked expression of IL-6 and IL-10 within hours of antigen uptake, suggesting that resident tissue APCs have the capacity to promote Th2 T cell differentiation in situ.

Role of genetic background in P selectin-dependent immune surveillance of the central nervous system.

Although the blood-brain barrier and blood cerebrospinal fluid barrier maintain the central nervous system (CNS) as an immunologically privileged site, T lymphocytes can migrate through unstimulated brain endothelium and epithelium to perform immune surveillance or initiate inflammation. Our prior results suggested that early CNS migration of a CD4 Th1 cell line was facilitated by P selectin (CD62P) in (PL/JxSJL/J)F1 mice. Here, quantitative analysis of migration 2 h following adoptive transfer of fluorescently labeled cells revealed a 53-72% decrease in activated splenocyte, CD4 Th1 and CD8 migration, but not CD4 Th2, in CD62P-deficient C57BL6/J mice. Immunohistochemistry revealed constitutive expression of CD62P within the meninges and choroid plexus epithelia in C57BL6/J and SJL/J, but not BALB/cJ, mice. Activated splenocyte migration was approximately three- to four-fold greater in SJL/J as compared to BALB/cJ mice. Anti-CD62P treatment normalized this difference. Based on these results, we hypothesize that genetically determined kinetics of immune surveillance may regulate the phenotype of subsequent CNS inflammation.