Orthotopic mouse models expressing fluorescent proteins for cancer drug discovery

Importance of the field: Currently used rodent tumor models, including transgenic tumor models, or subcutaneously growing human tumors in immunodeficient mice, do not sufficiently represent clinical cancer, especially with regard to metastasis and drug sensitivity.

Areas covered in this review: To obtain clinically accurate models, we have developed the technique of surgical orthotopic implantation (SOI) to transplant histologically intact fragments of human cancer, including tumors taken directly from the patient, to the corresponding organ of immunodeficient rodents. SOI allows the growth and metastatic potential of the transplanted tumors to be expressed and reflects clinical cancer of all types. Effective drugs can be discovered and evaluated in the SOI models utilizing human tumor cell lines and patient tumors. Visualization of many aspects of cancer initiation and progression in vivo has been achieved with fluorescent proteins. Tumors and metastases in the SOI models that express fluorescent proteins can be visualized noninvasively in intact animals, greatly facilitating drug discovery.

What the reader will gain: This review will provide information on the imageable mouse models of cancer that are clinically relevant, especially regarding metastasis and their use for drug discovery and evaluation.

Take home message: SOI mouse models of cancer reproduce the features of clinical cancer.     

Drug design strategies for the treatment of prostate cancer

Introduction: While metastatic prostate cancer remains an incurable tumor, remarkable progress has been made with novel drug design strategies for this incurable disease. Several new agents, including hormonal analogues, cytotoxic chemotherapy drugs, radionuclides and innovative targeted therapies, have recently been approved by the FDA for use in advanced and/or metastatic castrate-resistant prostate cancer. Furthermore, a growing number of new diagnostic or predictive genetic tests have also been incorporated into the management of this disease. Immunotherapy-based approaches have shown promise and have led to drug approvals. Other experimental approaches such as vascular targeting are in early translational clinical trials.

Areas covered: Herein, the authors outline select state-of-the-art approaches in the field. They also discuss the current challenges and future opportunities in the medical care of prostate cancer patients.

Expert opinion: An inherent challenge in the treatment of prostate cancer is to determine which patients need immediate aggressive treatment versus active surveillance. For patients needing aggressive treatment, integrating the sequence of therapeutic interventions, to provide the most benefit, remains a challenge that clinicians face. Recently, several genetic tests have been approved, facilitating early treatment decisions. Innovative targeted therapies are moving towards clinical applications, providing treatment options for tumors previously considered refractory to androgen ablation treatment.     

Orthotopic Metastatic Mouse Models for Anticancer Drug Discovery and Evaluation: a Bridge to the Clinic

Currently used rodent tumor models, including transgenictumor models, or subcutaneously-growing human tumors inimmunodeficient mice, do not sufficiently represent clinicalcancer, especially with regard to metastasis and drugsensitivity. In order to obtain clinically accurate models, wehave developed the technique of surgical orthotopic implantation(SOI) to transplant histologically-intact fragments of humancancer, including tumors taken directly from the patient, to thecorresponding organ of immunodeficient rodents. It has beendemonstrated in 70 publications describing 10 tumor types thatSOI allows the growth and metastatic potential of thetransplanted tumors to be expressed and reflects clinical cancer.Unique clinically-accurate and relevant SOI models of humancancer for antitumor and antimetastatic drug discovery include:spontaneous SOI bone metastatic models of prostate cancer, breastcancer and lung cancer; spontaneous SOI liver and lymph nodeultra-metastatic model of colon cancer, metastatic models ofpancreatic, stomach, ovarian, bladder and kidney cancer.Comparison of the SOI models with transgenic mouse models ofcancer indicate that the SOI models have more features ofclinical metastatic cancer. Cancer cell lines have been stablytransfected with the jellyfish Aequorea victoriagreenfluorescent protein (GFP) in order to track metastases in freshtissue at ultra-high resolution and externally image metastasesin the SOI models. Effective drugs can be discovered andevaluated in the SOI models utilizing human tumor cell lines andpatient tumors. These unique SOI models have been used forinnovative drug discovery and mechanism studies and serve as abridge linking pre-clinical and clinical research and drugdevelopment.     

Animal models of skin disease for drug discovery

Introduction: Discovery of novel drugs, treatments, and testing of consumer products in the field of dermatology is a multi-billion dollar business. Due to the distressing nature of many dermatological diseases, and the enormous consumer demand for products to reverse the effects of skin photodamage, aging, and hair loss, this is a very active field.

Areas covered: In this paper, we will cover the use of animal models that have been reported to recapitulate to a greater or lesser extent the features of human dermatological disease. There has been a remarkable increase in the number and variety of transgenic mouse models in recent years, and the basic strategy for constructing them is outlined.

Expert opinion: Inflammatory and autoimmune skin diseases are all represented by a range of mouse models both transgenic and normal. Skin cancer is mainly studied in mice and fish. Wound healing is studied in a wider range of animal species, and skin infections such as acne and leprosy also have been studied in animal models. Moving to the more consumer-oriented area of dermatology, there are models for studying the harmful effect of sunlight on the skin, and testing of sunscreens, and several different animal models of hair loss or alopecia.     

Colorectal cancer models for novel drug discovery

Introduction: Despite increased screening rates and advances in targeted therapy, colorectal cancer (CRC) remains the third leading cause of cancer-related mortality. CRC models that recapitulate key features of human disease are essential to the development of novel and effective therapeutics. Classic methods of modeling CRC such as human cell lines and xenograft mice, while useful for many applications, carry significant limitations. Recently developed in vitro and in vivo models overcome some of these deficiencies and thus can be utilized to better model CRC for mechanistic and translational research.

Areas covered: The authors review established models of in vitro cell culture and describe advances in organoid culture for studying normal and malignant intestine. They also discuss key features of classic xenograft models and describe other approaches for in vivo CRC research, including patient-derived xenograft, carcinogen-induced, orthotopic transplantation and transgenic mouse models. We also describe mouse models of metastatic CRC.

Expert opinion: No single model is optimal for drug discovery in CRC. Genetically engineered models overcome many limitations of xenograft models. Three-dimensional organoids can be efficiently derived from both normal and malignant tissue for large-scale in vitro and in vivo (transplantation) studies and are thus a significant advance in CRC drug discovery.     

Zibotentan for the treatment of castrate-resistant prostate cancer

Importance of the field: Patients with prostate cancer who have progression of their disease while on androgen deprivation therapy have limited therapeutic options. Docetaxel is currently the only agent that increases overall survival in patients with metastatic, castration-resistant prostate cancer; additional agents are needed.

Areas covered in this review: This review will describe the importance of endothelin-1 (ET-1) for growth of prostate cancer cells, development of bone metastases, and pain responses; the preclinical data for zibotentan, a specific inhibitor of the ET_A receptor; and the clinical development of atrasentan, a first-generation ET receptor inhibitor, and zibotentan, a more selective inhibitor of the ET_A receptor.

What the reader will gain: Readers will understand the importance of ET-1 as a novel pathway to target for patients with castration-resistant prostate cancer due to its association with prostate cancer growth, metastases to bone, and pain. Readers will learn about the preclinical and clinical development of zibotentan, including the promising Phase II results that have resulted in an extensive Phase III clinical trials program.

Take home message: Modulating the activity of ET-1 through the ET_A receptor is a novel target for treating patients with metastatic, castration-resistant prostate cancer. There are currently three ongoing Phase III trials with zibotentan, a selective ET_A inhibitor, to determine the effect of this agent on overall survival in these patients.     

Kallikrein-related peptidases targeted therapies in prostate cancer: perspectives and challenges

Introduction: Despite the emergence of several new effective treatments for metastatic castration-resistant prostate cancer patients, disease progression inevitably occurs, leading scientific community to carefully look for novel therapeutic targets of prostate cancer. Kallikrein (KLK)-related peptidases have been demonstrated to facilitate prostate tumorigenesis and disease progression through the development of an oncogenic microenvironment for prostate cells.

Areas covered: This review first summarizes the large amount of preclinical data showing the involvement of KLKs in prostate cancer pathobiology. In the second part, the authors assess the current status and future directions for KLK-targeted therapy and briefly describe the advances and challenges implicated in the design of effective manufactured drugs. The authors then focus on the preclinical data and on Phase I/II studies of the most promising KLK-targeted agents in prostate cancer. The drugs discussed here are divided on the basis of their mechanism of action: KLK-engineered inhibitors; KLK-activated pro-drugs; KLK-targeted microRNAs and small interfering RNAs^-/small hairpin RNAs; KLK vaccines and antibodies.

Expert opinion: Targeting KLK expression and/or activity could be a promising direction in prostate cancer treatment. Future human clinical trials will help us to evaluate the real benefits, toxicities and the consequent optimal use of KLK-targeted drugs, as mono-therapy or in combination regimens.     

Angiogenesis inhibitors in the treatment of prostate cancer

Importance of the field: Prostate carcinoma is the most common non-cutaneous malignancy in U.S. men. The efficacy of docetaxel and prednisone in metastatic castrate-resistant prostate cancer (mCRPC) has been shown to improve overall survival; however, its effect is not durable, highlighting the need for new therapies.

Areas covered in this review: We will review the development of some of the leading compounds with direct and indirect antiangiogenic activity in prostate cancer including antibodies to VEGF and its receptors, small-molecule inhibitors of downstream signaling, immunomodulatory drugs with antiangiogenic activity, and compounds thought to directly inhibit or destroy vascular endothelial cells.

What the reader will gain: The reader will gain a basic understanding of the role of angiogenesis in prostate cancer growth and metastasis. Current and potential targets of angiogenesis and their corresponding drugs under development for prostate cancer are discussed.

Take home message: There are now multiple early-phase clinical trials of antiangiogenic agents alone or in combination in prostate cancer. Several of these agents are now in Phase III development. Combined therapy with two antiangiogenic compounds may improve the activity of either compound alone. Multiple targets in the angiogenesis pathway continue to be elucidated and should remain an active area of investigation for the treatment of prostate cancer.     

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver

Abstract

1.?Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans.

2.?Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice.

3.?Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans.

4.?Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans.

5.?The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.     

Zebrafish xenograft models of cancer and metastasis for drug discovery

Introduction: Patients with metastatic cancer suffer the highest rate of cancer-related death, but existing animal models of metastasis have disadvantages that limit our ability to understand this process. The zebrafish is increasingly used for cancer modelling, particularly xenografting of human cancer cell lines, and drug discovery, and may provide novel scientific and therapeutic insights. However, this model system remains underexploited.

Areas covered: The authors discuss the advantages and disadvantages of the zebrafish xenograft model for the study of cancer, metastasis and drug discovery. They summarise previous work investigating the metastatic cascade, such as tumour-induced angiogenesis, intravasation, extravasation, dissemination and homing, invasion at secondary sites, assessing metastatic potential and evaluation of cancer stem cells in zebrafish.

Expert opinion: The practical advantages of zebrafish for basic biological study and drug discovery are indisputable. However, their ability to sufficiently reproduce and predict the behaviour of human cancer and metastasis remains unproven. For this to be resolved, novel mechanisms must to be discovered in zebrafish that are subsequently validated in humans, and for therapeutic interventions that modulate cancer favourably in zebrafish to successfully translate to human clinical studies. In the meantime, more work is required to establish the most informative methods in zebrafish.     

Update on apelin peptides as putative targets for cardiovascular drug discovery

Introduction: The physiological importance of GPCR/ligand pathways is highlighted by the fact that numerous pathologies are attributed to their signaling dysfunction. Over 50% of the pharmaceutical drugs currently used to treat human disease are based on compounds that interact with GPCRs. Apelin/APJ constitutes a novel endogenous peptide/GPCR system proposed to be involved in a wide range of physiological functions. Early evidence suggests that apelin/APJ may hold promise as a target for development of novel therapeutic agents which may counteract a number of pathologies including cardiovascular disease. Despite advances in treatment of cardiovascular disease, incidence, prevalence, morbidity and economic costs remain high necessitating the development of new treatment paradigms.

Areas covered: This review summarizes apelin/APJ structure, distribution and regulation; presents evidence for a role of apelin in pressure/volume homeostasis and in the pathophysiology of cardiovascular disease; summarizes data on beneficial effects of apelin in preclinical, animal models of cardiovascular disease and measurement of plasma levels of apelin across the full spectrum of cardiovascular disease in humans; and notes the first studies describing bioactivity of apelin peptides in human healthy volunteers and patients with heart failure.

Expert opinion: More clarity is needed on the precise physiological/pathophysiological role of the apelin/APJ system in human health and disease. Nonetheless, preclinical studies and initial studies in humans show that APJ antagonism may represent a novel therapeutic target for patients with cardiovascular disease. Development of appropriately validated assays for apelin will clarify circulating levels of the peptide in health and disease. Development of suitable agonists/antagonists will pave the way for much needed future studies essential for advancing this promising field of drug discovery.     

The hurdle of antiandrogen drug resistance: drug design strategies

Introduction: Prostate cancer is the second most common cancer death in men after lung cancer, due to distant metastases. While distant prostate cancer is typically castrate resistant, it is not necessarily androgen independent. For this reason, a review of the literature regarding the pathways involved in androgen signaling and therapeutic regimens to treat distant metastases is beneficial to increasing the survival rate of prostate cancer patients.

Areas covered: In this article, the author reviews the literature from the past decade covering metastatic hormone refractory prostate cancer with the aim to examine and identify pathways, therapeutic targets and current therapies for treating castrate-resistant disease. As this area is lacking, the author aims to provide the reader with knowledge of the molecular consequences of castrate resistant prostate cancer, the current treatment paradigms and future directions.

Expert opinion: While there have been advances in the treatment of castrate resistant prostate cancer, only minimal advances have been made in overall survival rate. Due to aberrant mutations and activation in the androgen receptor gene, and the complexity of cell signaling within prostate cancer, the androgen receptor should remain a main target for drug discovery efforts. This author believes that designing compounds that will reduce the activation of the androgen receptor may hold the key to a cure in the future.     

Emerging drugs for prostate cancer

Introduction: Androgen deprivation therapy is the mainstay treatment for patients with prostate cancer who are not candidates for definitive treatment, are diagnosed with advanced disease on initial presentation or progress after primary treatment. Patients who stop responding to androgen deprivation therapy develop castration resistant prostate cancer (CRPC). Emerging drugs undergoing clinical evaluation and drugs that have recently received FDA approval for the treatment of CRPC are reviewed.

Areas covered: As the natural history and signaling pathways of prostate cancer are better understood, new treatments and targeted therapies will be developed. The FDA recently approved 5 medications that increase survival in patients with CRPC. Additional medications and drug classes are being explored that may eventually lead to new treatment options. Articles were identified using a PubMed database search.

Expert opinion: Recent FDA medication approvals and the development of emerging treatments are promising for the future of patients with prostate cancer. The addition of new medications challenges physicians to identify the optimal sequence and/or combination in which newer and older medications should be administered. Physicians treating patients with prostate cancer have a growing responsibility to keep pace with these new medications so that they may counsel and treat patients appropriately.     

Androgen pathway resistance in prostate cancer and therapeutic implications

Introduction: Metastatic prostate cancer is an incurable disease that is treated with a variety of hormonal therapies targeting various nodes of the androgen receptor (AR) pathway. Invariably patients develop resistance and become castration resistant. Common treatments for castration-resistant disease include novel hormonal therapies, such as abiraterone and enzalutamide, chemotherapy, immunotherapy and radiopharmaceuticals. As this disease generally remains incurable, understanding the molecular underpinnings of resistance pathways is critical in designing therapeutic strategies to delay or overcome such resistance.

Areas covered: This review will explore the resistance mechanisms relevant to hormonal agents, such as AR-V7 expression and others, as well as discussing new approaches being developed to treat patients with castration-resistant prostate cancer that take advantage of these new insights. A literature search was performed to identify all published clinical trials related to androgen therapy mechanisms of drug resistance in metastatic castration-resistant prostate cancer.

Expert opinion: Androgen therapy resistance mechanisms are varied, and include modification of all nodes in the androgen signaling pathway. The optimal treatment for men with relapsed metastatic castration-resistant prostate cancer is uncertain at this time. The authors recommend using available clinical data to guide treatment decision making until more specific biomarkers are clinically available.     

Modeling therapy resistance in genetically engineered mouse cancer models

Resistance to anti-cancer drugs is a major obstacle in successful treatment of cancer. Multidrug resistance is not only observed with clinically established chemotherapeutics, but also with novel targeted therapies. Although a range of drug resistance mechanisms have been identified up till now, for most drugs it is still controversial which mechanisms are responsible for resistance and therapy failure in patients. Hence, the development of strategies to circumvent drug resistance is often unfocused. Since several years genetically engineered mouse models have been generated which develop tumors that closely resemble cancer in humans. We argue that such models can be used to investigate relevant in vivo mechanisms of resistance. This includes the analysis of intrinsic and acquired resistance, and the characterization of residual cells which survive the treatment. In such model systems different drugs and therapy combinations can be optimized prior to clinical trials.     

Drug metabolism by flavin-containing monooxygenases of human and mouse

Introduction: Flavin-containing monooxygenases (FMOs) play an important role in drug metabolism.

Areas covered: We focus on the role of FMOs in the metabolism of drugs in human and mouse. We describe FMO genes and proteins of human and mouse; the catalytic mechanism of FMOs and their significance for drug metabolism; differences between FMOs and CYPs; factors contributing to potential underestimation of the contribution of FMOs to drug metabolism; the developmental and tissue-specific expression of FMO genes and differences between human and mouse; and factors that induce or inhibit FMOs. We discuss the contribution of FMOs of human and mouse to the metabolism of drugs and how genetic variation of FMOs affects drug metabolism. Finally, we discuss the utility of animal models for FMO-mediated drug metabolism in humans.

Expert opinion: The contribution of FMOs to drug metabolism may be underestimated. As FMOs are not readily induced or inhibited and their reactions are generally detoxifications, the design of drugs that are metabolized predominantly by FMOs offers clinical advantages. Fmo1^(-/-),Fmo2^(-/-),Fmo4^(-/-) mice provide a good animal model for FMO-mediated drug metabolism in humans. Identification of roles for FMO1 and FMO5 in endogenous metabolism has implications for drug therapy and initiates an exciting area of research.     

Advancing therapies in metastatic castration-resistant prostate cancer

ABSTRACT

Introduction: Prostate cancer is the second most common cause of cancer worldwide and is the most frequently detected cancer in the European Union in men over 50 years of age. Androgen deprivation therapy remains the cornerstone of treatment for recurrent or metastatic disease. Unfortunately, nearly all patients will develop resistance to androgen blockade leading to castration-resistant prostate cancer (CRPC). Over the last 10 years, new treatments have dramatically improved overall survival of men with mCRPC. Current therapies are based on AR-axis inhibitors and taxane-based chemotherapies, as well as radiopharmaceuticals and Sipuleucel T.

Areas covered: The authors provide a review of the current field of systemic therapy in metastatic CRPC. This is followed by an in-depth analysis of recent developments in treatment, and the biological rationale behind these therapies.

Expert opinion: Since several trials with docetaxel or novel hormonal agents showed improvement in overall survival in metastatic castration-sensitive prostate cancer, as well as in non-metastatic castration-resistant patients, it is expected that a growing subgroup of patients will be exposed earlier to chemotherapy and to AR targeted agents. It becomes then fundamental to find novel strategies to overcome drug resistance and further improve survival.     

Src kinase inhibitors: an emerging therapeutic treatment option for prostate cancer

Importance of the field: Once prostate cancer becomes castration-resistant, bone metastases are a significant problem and treatment options are limited. As a result, there is a need for more effective therapies that have antitumor and anti-bone metastatic effects. Because Src and Src-family kinases (SFKs) are involved in multiple signaling pathways central to prostate cancer development, progression, and metastasis, in addition to normal and pathologic osteoclast activities, Src inhibition represents a valid therapeutic strategy for investigation.

Areas covered in this review: Here, current treatment options for advanced prostate cancer, the preclinical rationale behind using Src inhibitors, emerging data from clinical trials of Src inhibitors in prostate cancer, and future therapeutic directions are described. Data published in peer-reviewed journals within the last 20 years or presented at recent European or American Society of Clinical Oncology conferences have been reviewed.

What the reader will gain: Readers will gain an insight into the development of therapeutic Src inhibitors, including dasatinib and saracatinib; an understanding of their effects on prostate cancer cells and the bone microenvironment; and emerging clinical data.

Take home message: Src is implicated in prostate cancer progression and metastasis, therefore treatment with Src inhibitors warrants further investigation.     

Targeting the 5′-AMP-activated protein kinase and related metabolic pathways for the treatment of prostate cancer

Introduction: Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5′-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer.

Areas covered: Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling.

Expert opinion: Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.