Resistance in the anti-angiogenic era: nay-saying or a word of caution?

The hope that anti-angiogenic therapy might be the panacea for cancer has not yet been realized. There are many possible reasons for this, including endothelial and tumor cell heterogeneity, the presence of survival factors within the tumor micro-environment, the problem of defining the best dose and schedule for anti-angiogenic therapies, and angiogenesis-independent regrowth of tumors. Once these problems are understood, we can begin to evaluate approaches to thwarting them. These could include combining anti-angiogenic therapies with chemotherapy, with other anti-angiogenic agents or with other biological therapies. Another approach would be to employ a particular agent or combination of agents that target processes crucial to the survival of a particular cancer.     

Selective blockade of tumor angiogenesis

Blocking tumor angiogenesis is an important goal of cancer therapy, but clinically approved anti-angiogenic agents suffer from limited efficacy and adverse side effects, fueling the need to identify alternative angiogenesis regulators. Tumor endothelial marker 8 (TEM8) is a highly conserved cell surface receptor overexpressed on human tumor vasculature. Genetic disruption of Tem8 in mice revealed that TEM8 is important for promoting tumor angiogenesis and tumor growth but dispensable for normal development and wound healing. The induction of TEM8 in cultured endothelial cells by nutrient or growth factor deprivation suggests that TEM8 may be part of a survival response pathway that is activated by tumor microenvironmental stress. In preclinical studies, antibodies targeted against the extracellular domain of TEM8 inhibited tumor angiogenesis and blocked the growth of multiple human tumor xenografts. Anti-TEM8 antibodies augmented the activity of other anti-angiogenic agents, vascular targeting agents and conventional chemotherapeutic agents and displayed no detectable toxicity. Thus, anti-TEM8 antibodies provide a promising new tool for selective blockade of neovascularization associated with cancer and possibly other angiogenesis-dependent diseases.     

Selective blockade of tumor angiogenesis

Blocking tumor angiogenesis is an important goal of cancer therapy, but clinically approved anti-angiogenic agents suffer from limited efficacy and adverse side effects, fueling the need to identify alternative angiogenesis regulators. Tumor endothelial marker 8 (TEM8) is a highly conserved cell surface receptor overexpressed on human tumor vasculature. Genetic disruption of Tem8 in mice revealed that TEM8 is important for promoting tumor angiogenesis and tumor growth but dispensable for normal development and wound healing. The induction of TEM8 in cultured endothelial cells by nutrient or growth factor deprivation suggests that TEM8 may be part of a survival response pathway that is activated by tumor microenvironmental stress. In preclinical studies, antibodies targeted against the extracellular domain of TEM8 inhibited tumor angiogenesis and blocked the growth of multiple human tumor xenografts. Anti-TEM8 antibodies augmented the activity of other anti-angiogenic agents, vascular targeting agents and conventional chemotherapeutic agents and displayed no detectable toxicity. Thus, anti-TEM8 antibodies provide a promising new tool for selective blockade of neovascularization associated with cancer and possibly other angiogenesis-dependent diseases.     

Control of tumor-related angiogenesis]

Tumor-related angiogenes is expected to become an important target for improving treatment of cancer, because it plays key roles in tumor growth, invasion and metastasis. We considered that the successful development of such angiostatic treatment depended entirely upon the development of useful anti-angiogenic agents, and attempted to find novel angiogenesis inhibitors by using three in vivo assays, based on an idea of ours. As a result we have demonstrated that different types of agents with low molecular weight including microbial metabolites, cell differentiation modulators like retinoids and steroids, exhibit anti-angiogenic activity, anti-metastatic activity and/or antitumor activity. Taken these findings, an ideal anti-angiogenic agent is discussed.     

Angiogenesis in gynecological cancers and the options for anti-angiogenesis therapy

Angiogenesis is required in cancer, including gynecological cancers, for the growth of primary tumors and secondary metastases. Development of anti-angiogenesis therapy in gynecological cancers and improvement of its efficacy have been a major focus of fundamental and clinical research. However, survival benefits of current anti-angiogenic agents, such as bevacizumab, in patients with gynecological cancer, are modest. Therefore, a better understanding of angiogenesis and the tumor microenvironment in gynecological cancers is urgently needed to develop more effective anti-angiogenic therapies, either or not in combination with other therapeutic approaches. We describe the molecular aspects of (tumor) blood vessel formation and the tumor microenvironment and provide an extensive clinical overview of current anti-angiogenic therapies for gynecological cancers. We discuss the different phenotypes of angiogenic endothelial cells as potential therapeutic targets, strategies aimed at intervention in their metabolism, and approaches targeting their (inflammatory) tumor microenvironment.     

Androgen Receptor as a Driver of Therapeutic Resistance in Advanced Prostate Cancer

The role of the androgen receptor (AR) signaling axis in the progression of prostate cancer is a cornerstone to our understanding of the molecular mechanisms causing castration-resistant prostate cancer (CRPC). Resistance of advanced prostate cancer to available treatment options makes it a clinical challenge that results in approximately 30,000 deaths of American men every year. Since the historic discovery by Dr. Huggins more than 70 years ago, androgen deprivation therapy (ADT) has been the principal treatment for advanced prostate cancer. Initially, ADT induces apoptosis of androgen-dependent prostate cancer epithelial cells and regression of androgen-dependent tumors. However, the majority of patients with advanced prostate cancer progress and become refractory to ADT due to emergence of androgen-independent prostate cancer cells driven by aberrant AR activation. Microtubule-targeting agents such as taxanes, docetaxel and paclitaxel, have enjoyed success in the treatment of metastatic prostate cancer; although new, recently designed mitosis-specific agents, such as the polo-kinase and kinesin-inhibitors, have yielded clinically disappointing results. Docetaxel, as a first-line chemotherapy, improves prostate cancer patient survival by months, but tumor resistance to these therapeutic agents inevitably develops. On a molecular level, progression to CRPC is characterized by aberrant AR expression, de novo intraprostatic androgen production, and cross talk with other oncogenic pathways. Emerging evidence suggests that reactivation of epithelial-mesenchymal-transition (EMT) processes may facilitate the development of not only prostate cancer but also prostate cancer metastases. EMT is characterized by gain of mesenchymal characteristics and invasiveness accompanied by loss of cell polarity, with an increasing number of studies focusing on the direct involvement of androgen-AR signaling axis in EMT, tumor progression, and therapeutic resistance. In this article, we discuss the current knowledge of mechanisms via which the AR signaling drives therapeutic resistance in prostate cancer metastatic progression and the novel therapeutic interventions targeting AR in CRPC.     

Targeting vasculature in urologic tumors: mechanistic and therapeutic significance

Recent advances toward understanding the molecular mechanisms regulating cancer initiation and progression provide new insights into the therapeutic value of targeting tumor vascularity by interfering with angiogenic signaling pathways. The functional contribution of key angiogenic factors toward increased vascularity characterizing metastatic tumors and their therapeutic exploitation is considered in three major urologic malignancies, renal, bladder, and prostate cancer. With the realization that the success of the therapeutic efficacy of the various anti-angiogenic approaches for the treatment of urologic tumors has yet to be proven clinically, the challenge remains to select critical angiogenesis pathways that can be targeted for an individual tumor. Here we discuss the major mechanisms that support formation of vasculature in renal, bladder, and prostate tumors and the current results of targeting of specific molecules/regulators for therapeutic intervention against metastastic disease.     

Tumor vessel stabilization and remodeling by anti-angiogenic therapy with bevacizumab

Bevacizumab-resistant tumor vessels were characterized by an increased vessel diameter and normalization of vascular structures by the recruitment of mature pericytes and smooth muscle cells. Here, we analyzed human liver metastases which were taken at clinical relapse in patients with colorectal adenocarcinoma treated with anti-angiogenic therapy using the humanized monoclonal anti-VEGF bevacizumab. Tumor vessels which are resistant to anti-VEGF therapy are increased in size and characterized by a normalization of the vascular bed. These results were confirmed using NOD SCID mice as animal model and xenograft transplantation of human PC-3 prostate carcinoma cells in combination with bevacizumab treatment. Our results confirmed that anti-angiogenic therapy results in enhanced vascular remodeling by vascular stabilization. This process is apparently accompanied by enhanced necrosis of tumor tissue. These processes interfere with the efficacy of anti-angiogenic therapy because of reduced susceptibility of stabilized vessels by this therapy. These results demonstrate the importance for the development of second generation anti-angiogenic combination therapy concepts to rule out the balance between vascular stabilization followed by a possible de-stabilization making the remained vessels susceptible to a second wave of anti-angiogenic therapy.     

Epithelial mesenchymal transition and resistance in endocrine-related cancers

Epithelial to mesencyhmal transition (EMT) has a central role in tumor metastasis and progression. EMT is regulated by several growth factors and pro-inflammatory cytokines. The most important role in this regulation could be attributed to transforming growth factor-β (TGF-β). In breast cancer, TGF-β effect on EMT could be potentiated by Fos-related antigen, oncogene HER2, epidermal growth factor, or mitogen-activated protein kinase kinase 5 – extracellular-regulated kinase signaling. Several microRNAs in breast cancer have a considerable role either in potentiation or in suppression of EMT thus acting as oncogenic or tumor suppressive modulators. At present, possibilities to target EMT are discussed but the results of clinical translation are still limited. In prostate cancer, many cellular events are regulated by androgenic hormones. Different experimental results on androgenic stimulation or inhibition of EMT have been reported in the literature. Thus, a possibility that androgen ablation therapy leads to EMT thus facilitating tumor progression has to be discussed. Novel therapy agents, such as the anti-diabetic drug metformin or selective estrogen receptor modulator ormeloxifene were used in pre-clinical studies to inhibit EMT in prostate cancer. Taken together, the results of pre-clinical and clinical studies in breast cancer may be helpful in the process of drug development and identify potential risk during the early stage of that process.     

The quest for surrogate markers of angiogenesis: a paradigm for translational research in tumor angiogenesis and anti-angiogenesis trials

Inhibition of tumor angiogenesis suppresses tumor growth and metastatic spreading in many experimental models, suggesting that anti-angiogenic drugs may be used to treat human cancer. During the past decade more than eighty molecules that showed anti-angiogenic activity in preclinical studies were tested in clinical cancer trials, but most of them failed to demonstrate any measurable anti-tumor activity and none have been approved for clinical use. Recent results stemming from trials with anti-VEGF antibodies, used alone or in combination with chemotherapy, suggest that systemic anti-angiogenic therapy may indeed have a measurable impact on cancer progression and patient survival. From the clinical studies it became nevertheless clear that the classical endpoints used in anti-cancer trials do not bring sufficient discriminative power to monitor the effects of anti-angiogenic drugs. It is therefore necessary to identify and validate molecular, cellular and functional surrogate markers of angiogenesis to monitor activity and efficacy of anti-angiogenic drugs in patients. Availability of such markers will be instrumental to re-evaluate the role of tumor angiogenesis in human cancer, to identify new molecular targets and drugs, and to improve planning, monitoring and interpretation of future studies. Future anti-angiogenesis trials integrating biological endpoints and surrogate markers or angiogenesis will require close collaboration between clinical investigators and laboratory-based researchers.     

Use of transgenic mice as models for prostate cancer chemoprevention

Prostate cancer is a long latency type of tumor that usually develops in men older than 50 years of age. Prostate epithelial neoplasia (PIN), the initial malignant lesion, progresses to invasive carcinoma over the course of years. Because of the particular features of prostate carcinogenesis, this type of tumor may represent a paradigm for cancer prevention. Several clinical trials have evaluated the effect of different compounds on prostate tumor development, including finasteride, selenium, vitamin E, and carotenes. Although some results are promising, no conclusive data have been achieved as to recommend any of these compounds as preventive agents. Results from some trials, such as SELECT, where supplementation of selenium and/or vitamin-E was used, have been rather disappointing. However, many novel chemopreventive agents that target different cancer-related pathways are being developed lately. Appropriate animal models (in particular, genetically modified mice) are being used to assess the efficacy of these novel compounds. The transgenic adenocarcinoma of the mouse prostate (TRAMP) model has been validated as an accurate model to test a variety of preventive agents. Genistein, alpha-difluoromethylornithine, toremifene, R-flurbiprofen, celecoxib, and green tea polyphenols have been shown to prevent prostate cancer development in TRAMP mice. In conclusion, new chemopreventive compounds which are effective in animal models are likely to be tested soon in clinical trials, with the final goal of reducing prostate cancer incidence in men.     

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.     

JQ1, a BET‐bromodomain inhibitor,inhibits human cancer growth and suppresses PD‐L1 expression

Most traditional cytotoxic chemotherapeutic agents have poor aqueous solubility and significant toxicity. Hence, there is a need to develop molecule‐targeted drugs. Programmed death‐ligand 1 (PD‐L1) is associated with the prognosis of several cancer types, and blockade of PD‐1/PD‐L1 signaling increases the amplitude of anti‐tumor immunity. In the present study, we investigated the effects of JQ1, a bromodomain and extraterminal‐bromodomain inhibitor, on cell growth, and messenger RNA (mRNA) and protein levels of PD‐L1 in renal cell carcinoma primary culture cells, and prostate, liver, and lung cancer cell lines. The results of the cell counting kit‐8 assay suggested that JQ1 inhibits cell growth in a dose‐dependent manner. The mRNA and protein levels of PD‐L1 decreased in the primary culture of JQ1‐treated renal carcinoma, prostate cancer, liver cancer, and lung cancer cell lines. In addition, the mRNA level of PD‐L2 also decreased in the JQ1‐treated cells. Overall, JQ1 might be a potential anti‐tumor agent.     

A natural small molecule voacangine inhibits angiogenesis both in vitro and in vivo

Angiogenesis, the formation of new blood vessels from pre-existing ones, plays a critical role in normal and pathological phenotypes, including solid tumor growth and metastasis. Accordingly, the development of new anti-angiogenic agents is considered an efficient strategy for the treatment of cancer and other human diseases linked with angiogenesis. We have identified voacangine, isolated from Voacanga africana, as a novel anti-angiogenic agent. Voacangine inhibits the proliferation of HUVECs at an IC(50) of 18 μM with no cytotoxic effects. Voacangine significantly suppressed in vitro angiogenesis, such as VEGF-induced tube formation and chemoinvasion. Moreover, the compound inhibits in vivo angiogenesis in the chorioallantoic membrane at non-toxic doses. In addition, voacangine decreased the expression levels of hypoxia inducible factor-1α and its target gene, VEGF, in a dose-dependent manner. Taken together, these results suggest that the naturally occurring compound, voacangine, is a novel anti-angiogenic compound.     

Anti-angiogenic potential of a cancer chemopreventive flavonoid antioxidant, silymarin: inhibition of key attributes of vascular endothelial cells and angiogenic cytokine secretion by cancer epithelial cells

In recent studies, we have shown that silymarin, a naturally occurring flavonoid antioxidant, exhibits anti-cancer effects against several epithelial cancers. Here, we assessed its potential as an anti-angiogenic agent employing human umbilical vein endothelial cells (HUVEC) and human prostate and breast cancer epithelial cells. When sub-confluent HUVEC were treated for 48 h, adherent cell number decreased by 50 and 90% at 50 and 100 microg/ml doses, respectively. Apoptotic cell death principally accounted for cell loss at >50 microg/ml doses. In biochemical analysis, silymarin treatment of HUVEC for 6 h resulted in a concentration-dependent decrease in the secretion and cellular content of matrix metalloproteinase (MMP)-2/gelatinase A. Silymarin also inhibited HUVEC tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel. In other studies, 5 to 6 h exposure of DU145 prostate, and MCF-7 and MDA-MB-468 breast cancer cells to silymarin resulted in a dose-dependent decrease in the secreted vascular endothelial growth factor (VEGF) level in conditioned media without any visible change in cell morphology. The inhibitory effect of silymarin on VEGF secretion occurred as early as 1 h. These observations indicate a rapid inhibitory action of silymarin on the secretion of this primary angiogenic cytokine by cancer epithelial cells. Taken together, the results of this study support the hypothesis that silymarin possesses an anti-angiogenic potential that may critically contribute to its cancer chemopreventive efficacy.     

Lectin-Like Oxidized LDL Receptor-1 Is an Enhancer of Tumor Angiogenesis in Human Prostate Cancer Cells

Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells.     

Vitamin D and prostate cancer

Vitamin D and its metabolites are best known for their actions in calcium and bone metabolism. However, epidemiological studies have suggested that an increased prostate cancer risk is associated with decreased production of vitamin D. In vitro and in vivo studies have shown that the biologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25D), inhibits proliferation of cancer cells derived from multiple tissues, including the prostate. Although the mechanisms underlying the growth inhibitory effects of 1,25D have not been fully elucidated, in prostate cancer cells 1,25D reduces cell growth via a number of cellular pathways, including cell cycle arrest, induction of apoptosis, and altered activation of growth factor signaling. The hypercalcemia induced by 1,25D in vivo limits its use clinically as a therapeutic agent. However, several 1,25D analogs have been developed that reduce prostate tumor growth in rodent xenograft models without causing hypercalcemia. Additional studies are required in order to determine whether these 1,25D analogs will be useful therapeutic agents for the treatment of prostate cancer.     

Prostate cancer vaccines: the long road to clinical application

Cancer vaccines as a modality of immune-based cancer treatment offer the promise of a non-toxic and efficacious therapeutic alternative for patients. Emerging data suggest that response to vaccination largely depends on the magnitude of the type I immune response generated, epitope spreading and immunogenic modulation of the tumor. Moreover, accumulating evidence suggests that cancer vaccines will likely induce better results in patients with low tumor burden and less aggressive disease. To induce long-lasting clinical responses, vaccines will need to be combined with immunoregulatory agents to overcome tumor-related immune suppression. Immunotherapy, as a treatment modality for prostate cancer, has received significant attention in the past few years. The most intriguing characteristics that make prostate cancer a preferred target for immune-based treatments are (1) its relative indolence which allows sufficient time for the immune system to develop meaningful antitumor responses; (2) prostate tumor-associated antigens are mainly tissue-lineage antigens, and thus, antitumor responses will preferentially target prostate cancer cells. But, also in the event of eradication of normal prostate epithelium as a result of immune attack, this will have no clinical consequences because the prostate gland is not a vital organ; (3) the use of prostate-specific antigen for early detection of recurrent disease allows for the initiation of vaccine immunotherapy while tumor burden is still minimal. Finally, for improving clinical outcome further to increasing vaccine potency, it is imperative to recognize prognostic and predictive biomarkers of clinical benefit that may guide to select the therapeutic strategies for patients most likely to gain benefit.     

Anibamine,a natural product CCR5 antagonist,as a novel lead for the development of anti-prostate cancer agents

Accumulating evidence indicates that the chemokine receptor CCR5 and the chemokine CCL5 may be involved in the proliferation and metastasis of prostate cancer. Consequently, chemokine receptor CCR5 antagonists could potentially act as anti-prostate cancer agents. As the first natural product CCR5 antagonist, anibamine provides a novel chemical structural skeleton compared with other known antagonists identified through high-throughput screening. Our studies demonstrate that anibamine produces significant inhibition of prostate cancer cell proliferation at micromolar to submicromolar concentrations as well as suppressing adhesion and invasion of the highly metastatic M12 prostate cancer cell line. Preliminary in vivo studies indicate that anibamine also inhibits prostate tumor growth in mice. These findings indicate that anibamine may prove to be a novel lead compound for the development of prostate cancer therapeutic agents.