Unraveling the Complexities of Metastasis: Transition from a Segmented View to a Conceptual Continuum

Unraveling the Complexities of Metastasis: Transition from a Segmented View to a Conceptual Continuum provides a critical overview of the recent developments of metastasis research and how progress can be further enhanced in the field. Metastasis is a highly complicated mechanism and prognostic analysis of different metastatic patterns in advanced cancer patients is becoming increasingly problematic. It is therefore essential to take a step back and focus on the underlying mechanisms of metastasis before moving ahead for effective translation of laboratory findings to clinically effective therapeutics. This book is surely helpful in putting together missing pieces of an incomplete jig-saw puzzle of molecular cancer.

The book discusses topics such as the role of TRAIL-mediated signaling, late metastasis and mechanisms underlying tumor cell dormancy, CTCs and exomes, non-coding way of metastasis, and stem cells. Additionally, it brings relevant and updated information on nanotechnology-based docetaxel and the peculiarities of cancer cell metabolism.

This book is a valuable source for cancer researchers, medical doctors and several members of biomedical field who need to understand better the complex mechanism of metastasis.

• Explains the mechanism of metastasis from basic to advanced level through easy and comprehensive chapters written by internationally distinguished researchers
• Provides simplified version of important process of metastasis for the readers to comprehend the latest advancements made in the field
• Presents colorful diagrams to make different aspects of scientifically difficult topics easier for young researchers and newcomers in the field of cancer metastasis

• Language: English
• Publisher: Academic Press
• Release date: May 14, 2022
• ISBN: 9780128217900

Book preview

Preface

Cancer is a multifaceted, multistep, and therapeutically challenging disease. The heterogeneity of subpopulation of cancer cells is not only rooted in genetic variations and genomic instability within tumors but also within the intrinsic features of malignant progenitor cells for wide-ranging phenotypic variations. Increasingly, it is being realized that cancer cells disseminate from their primary original sites, intravasate into the blood circulation and lymphatic system, and finally extravasate to invade and colonize distant sites. Intriguingly, phenotypic plasticity allows tumor cells to rewire and adjust to specified microenvironments, tactfully overcome the challenging barriers of metastasis, and resist the effects of therapeutic combinations.

Integration of genomic, epigenomic, proteomic, and transcriptomic data has enabled us to refine our knowledge about malignant transformation, metastatic spread, and therapeutic outcomes.

Phenomenal theory proposed by Stephen Paget has revolutionized our understanding about the mechanisms which underpin metastasis. Landmark discoveries comprehensively unveiled protein networks and signaling pathways which propelled this exciting and sizzling field of research forward and brought metastasis biology to the forefront of clinical practice.

Conceptual realization of biological variability between micro- and macrometastasis, targeting of vulnerabilities of the metastatically competent cancer cells, as well as hallmark features of metastatic tumor microenvironment, set strong foundation for treatment of metastasis.

In this book, we have attempted to gather chef-d'oeuvre of scientifically distinguished researchers and clinicians across the globe. We have given special attention to the fundamental role of different signaling pathways in metastasis. In the first chapter, Farooqi et al highlighted oncogenic and prometastatic role of TRAIL-mediated signaling. It is indeed very surprising, but existing evidence also underscores the darker side of TRAIL-mediated signaling in different cancers. In the next chapter, Dr. Chiara Martinelli gave a summary of the roles of circulating tumor cells and exosomes in cancer metastasis. Dr. Wensi Tao from the Miller School of Medicine of University of Miami explained the underlying mechanisms of metastatic prostate cancer and how we can pharmacologically target these pathways for an efficient treatment. Professor Rossana Berardi gave expert opinion about prognostic and predictive role of bone metastasis in NSCLC. Dr. Maria Luisa Gasparri and her coauthors comprehensively analyzed and summarized how breast cancer cells gradually invaded the brain for metastatic spread and colonization. Chapter gives exciting information about the clues which entice breast cancer cells to pursue brain-related trajectories. Dr. Bakiye Goker Bagca and coauthors shared viewpoints about the linchpin role of noncoding RNAs in metastasis. Professor Indu Pal discussed a very important topic about the challenges faced during treatment with docetaxel in breast cancer patients. Professor Indu Pal and coauthors suggested that nanotechnology-based delivery of docetaxel might be a solution to the problems faced during the management of breast cancer and its metastasis. Dr. Violante Di Donato with his team provided conceptual basics and mechanisms associated with metastasis in gynecological cancers. Chapters 9 and 10 deal with the role of noncoding RNAs in metastasis. Dr. Seher Yilmaz and coauthors and Dr. Humaira Naureen and coauthors provided an interesting summary of regulation of HSP90 by noncoding RNAs and the role of circular RNAs in metastasis, respectively. Dr. Laura C. Ceafalan and her team members provided excellent view point on the central role of CD36 in metastasis. Whereas Dr. Tania Spohr comprehensively discussed how loss of balance of developmental genes played the role in different steps of metastasis, Professor Yusuf Tutar from Turkey has given his thoughts on the role of heat shock proteins in metastasis. Dr. Humaira Naureen and coauthors wrote a very intriguing chapter about kisspeptin-mediated signaling and how it regulated metastasis. Dr. Yomna Khamis and Professor Waleed Arafat from Egypt highlighted the interplay between tumor microenvironment and metastasis. These aspects are very informative and valuable for clinicians. Moreover, we have three chapters which deal with metastasis-inhibitory roles of medicinally active molecules from natural sources. Chapters 16, 18, and 19 deal with nutrigenomics and provide crisp information for nutritionists. Dr. Javed Iqbal and his coauthors summarized healthy and medicinally beneficial role of hydroxycinnamic acids and their derivatives. Dr. Durr-e-shahwar Malik gave a summary of beneficial properties of curcumin and how curcumin inhibited metastasis in different cancers. Ammad Ahmad Farooqi wrote an interesting chapter about metastasis-inhibitory role of blueberries. Professor Ilhan Yaylim from Turkey provided a concise overview of the fundamental concepts about the role of SMURFs in metastasis. The chapter is indeed very attractive because of nice diagrammatic representations. Dr. Giovanna Revilla and coauthors discussed about central role of ATP-binding cassette transporter genes in the metastasis. Dr. Madeline Farmer and coauthors critically discussed about role of epigenetics in metastasis. The final chapter is authored by Dr. Naila Malkani and coauthors. This chapter deals with molecular mechanisms associated with Ets-related ERG and miRNAs in metastasis.

We would like to pay our sincere gratitude to Rafael Teixeira and Timothy Bennett for their unflinching support and trust. We hope this book will be very exciting and informative for young researchers and distinguished scientists in the field of oncology.

Ammad Ahmad Farooqi

Muhammad Zahid Qureshi

Uteuliyev Yerzhan Sabitaliyevich

Chapter 1: Role of TRAIL-mediated signaling as Jekyll and Hyde in metastasis

changing places, changing faces

Ammad Ahmad Farooqi ¹ , Iqra Mobeen ² , Rukset Attar ³ , and Gamze Tanriover ⁴       ¹ Department of Molecular Oncology, Institute of Biomedical and Genetic Engineering (IBGE), Islamabad, Pakistan      ² Khursheed Rashid Hospital Lahore, Lahore, Pakistan      ³ Department of Obstetrics and Gynecology, Yeditepe University, Istanbul, Turkey      ⁴ Department of Histology and Embryology, School of Medicine, Akdeniz University, Antalya, Turkey

Abstract

Because of therapeutically challenging and heterogeneous nature of cancer, researchers have always focused on identification of molecules having minimum off-target effects and maximum therapeutic benefits. In accordance with this approach, restoration of apoptosis in drug-resistant cancers has been extensively studied. TRAIL-mediated apoptosis has gained tremendous limelight because of its unique ability to differentially target cancer cells which substantiate its legacy as a unique therapeutically advantageous anticancer agent. However, high-throughput technologies not only uncovered apoptosis-inducing activity of TRAIL but also unveiled darker aspects of TRAIL-mediated signaling. In this chapter, we have attempted to provide prometastatic facet of TRAIL-mediated nonapoptotic signaling.

Keywords

Apoptosis; Cancer; Metastasis; Signaling; TRAIL

Introduction

Apart from the heterogeneity in the TRAIL-mediated cascade originating from antagonistic and agonistic cell surface receptors, emerging evidence over the last few years has started to scratch the surface of additional layer of dichotomy within the TRAIL signaling network in different cancers. Discovery of TRAIL-mediated signaling cascade was doubtlessly groundbreaking and gained momentum because of its extraordinary property of differential killing effects. However, branching trajectories originating from TRAIL/TRAIL-R signaling axis are highly complicated and portray diametrically opposite cascades. There has been an exponential growth in the research reports related to TRAIL-mediated signaling for cancer therapy but parallel research works also highlighted prometastatic effects of TRAIL-driven nonapoptotic signaling.

Phosphorylation of cofilin at serine residue is triggered by LIMK (LIM domain kinase). LIMK is activated by ROCK (rho-associated, coiled-coil containing protein kinase). Mutant K-Ras has been shown to switch TRAIL-mediated cascade to prometastatic mode. In the presence of K-Ras, TRAIL and CD95L stimulated invasive potential of colorectal cancer cells and hepatic metastasis [1]. More importantly, loss of mutant K-Ras reactivated apoptosis-inducing characteristics of CD95 and TRAIL receptors and severely impaired metastasizing potential of cancer cells. Intriguingly, RAF1 was critical for the prometastatic functions of CD95 for survival of cancer cells in the liver and for K-Ras–induced hepatic metastasis. Data clearly suggested that K-Ras and RAF1 caused suppression of ROCK/LIMK-induced cofilin phosphorylation. Overexpression of LIMK or ROCK enabled CD95L to induce apoptotic death in K-Ras–proficient cells and prevented metastasis. However, suppression of LIMK or ROCK in K-Ras–deficient cancer cells provided protection against apoptotic death [1].

In mutant PIK3CA-expressing cells, TRAIL and CD95L stimulated the activation of NF-κB (nuclear factor kappa B) [2]. Importantly, TRAIL-mediated activation of NF-κB induced transformation of mutant PIK3CA-expressing cells to an amoeboid-like morphology. Caspase-8–induced cleavage of ROCK1 (rho-associated, coiled-coil containing protein kinase 1) is a novel mechanism for acquisition of ameboid morphology and enhanced invasiveness in response to CD95L and TRAIL. Pharmacological inhibition of ROCK1 effectively inhibited CD95L and TRAIL-induced invasive potential of HCT116-PIK3CA–mutant cells [2].

TRAIL triggered an increase in the phosphorylated levels of ERK only in mutant K-Ras–expressing adenocarcinoma cell lines (Pal [3]). However, MEK inhibitor PD98059 considerably reduced migratory potential of mutant K-Ras–expressing adenocarcinoma cell lines (Pal [3]).

Our rapidly evolving knowledge related to the pathways involved in the immune escape of cancer cells has allowed us to unravel an imperative role of immunosuppressive checkpoint signaling axis, such as PD-1 (programmed cell death 1 receptor) and its ligand PDL1 (programmed cell death ligand 1). TRAIL has also been shown to stimulate the expression of PDL1. KYSE-150 cells are highly metastatic esophageal squamous carcinoma cells (Zhang [4]). Levels of p-ERK and p-STAT3 were found to be reduced in TRAIL knockdown KYSE-150 cells. PDL1 expression was downregulated in STAT3-silenced EC1 cells. Furthermore, a number of pulmonary metastatic nodules were reported to be significantly increased in mice transplanted with TRAIL overexpressing EC1 cells. Moreover, pulmonary metastasis was found to be reduced in mice inoculated with TRAIL knockdown KYSE-150 cells (Zhang [4]). Overall, it seems clear that TRAIL-mediated cascade may also activate STAT3-induced upregulation of PDL1.

TRAIL-R2 knockdown potently reduced the formation of macroscopic liver metastasis in mice orthotopically inoculated with TRAIL-R2–silenced PancTu-I cells (Miarka [5]). In contrast to the reduced number of macrometastasis, livers of mice inoculated with TRAIL-R2–silenced PancTu-I cells demonstrated significantly higher number of disseminated tumor cells and micrometastasis. TRAIL-R2 knockdown restrained the progression of small metastatic lesions in the liver and suppressed the proliferation of tumor cells, thus reducing the outgrowth of postsurgical macroscopic metastases (Miarka [5]).

TRAIL-R2 expression was noted to be higher in bone-homing variant of MDA-MB-231 (Fritsche [6]). Phosphorylated levels of Src (nonreceptor tyrosine kinase) and AKT were noted to be reduced in MDA-MB-231-BO (bone-homing variant). For analysis of the roleplay of TRAIL-R2 in the formation and progression of bone metastasis, TRAIL-R2–silenced bone-homing variants of MDA-MB-231 cancer cells were intracardially injected into SCID mice. TRAIL-R2 knockdown led to significant reduction in the rate of metastases. Importantly, mice injected with TRAIL-R2–silenced bone-homing variant demonstrated a substantial decline in the average number of tumors in each xenograft model alongwith a shrinkage in average tumor area. Notably, TRAIL-R2 inhibition severely impaired the ability of osteotropic breast cancer cells to trigger skeletal metastasis (Fritsche [6]).

There are some exciting pieces of evidence which suggest that surviving cancer cells serve as a major source of TRAIL-mediated secretion of the cytokines. TRAIL sensitive cells produced very low levels of CXCL1 and interleukin-8 in HCT116 cells, while stimulation of colorectal HCT116 (TRAIL-resistant) cancer cells caused significant secretion of CXCL1 and interleukin-8 (Hartwig [7]). Notably, FADD-deficient tumors had notably lower infiltration of CD11b+GR1+ cells with low CD206+ expression. It is relevant to mention that expression of CD11b, GR1, and CD206 has previously been shown to be connected with alternative activation of M2-like myeloid cells. These cells have characteristically unique tumor-promoting activity. Thus, FADD not only promoted the growth of lung tumors but also prompted the formation of a chemically diverse cytokine milieu that supported development of the tumor. Additionally, FADD potently enhanced the infiltration of M2-like myeloid cells. Levels of interleukin-8, CCL2, and CXCL1 were reported to be reduced in the lungs of mice inoculated with FADD-deficient cancer cells. CCL2-CCR2 signaling axis is implicated in metastasis. Tumor burden was not to be considerably reduced in CCR2-deficient mice injected with TRAIL-R–silenced 3LL cells. Collectively, these findings clearly revealed that TRAIL-R expression is necessary for the production of CCL2 by tumor cells, which sequentially exerts protumorigenic effects through CCR2 present on the surface of host cells. Therefore, tumor cell–derived CCL2 failed to increase the infiltration rate of alternatively activated myeloid cells in the absence of host CCR2 (Hartwig [7]).

Darker side of caspase-8: nonapoptotic caspase-8–mediated functionalities

TRAIL and CD95L induced NF-κB, ERKs, JNK, and p38 in BCL-XL–overexpressing Colo357 cells (Siegmund [8]). zVAD-FMK is a pan-caspase inhibitor and robustly prevents apoptotic death in various cancer cell lines. Therefore, inhibition of caspases with zVAD-FMK potently blocked all these signaling pathways in BCL-XL–overexpressing Colo357 cells. zVAD-FMK considerably reduced CD95L and TRAIL-induced IκBα phosphorylation in BCL-XL–overexpressing Colo357 cells. Phosphorylation of IκBα relieved the inhibitory effects of IκBα on NF-κB. Caspase-triggered activation of NF-κB and ERK played an essential role in CD95L and TRAIL-induced upregulation of proinflammatory genes in BCL-XL–overexpressing Colo357 cells (Siegmund [8]). Collectively, these findings suggested that initiation of prosurvival signaling mainly through activation of NF-κB played linchpin role in enhanced survival rate and metastasizing potential of drug-resistant cancer cells.

TRAIL-mediated NF-κB activation: friend or foe

TRAIL-mediated signaling has been shown to activate NF-ҝB in different cancers. NF-ҝB activation adds new layer of intricacy to already complicated nature of TRAIL-mediated nonapoptotic signaling.

TRAIL-DR5 interaction activated NF-κB pathway in B16F10 cells [9]. TRAIL receptor activation by agonistic anti-DR5 mAb greatly increased the metastatic colonization of B16F10 cells in tumor-bearing mice (Takahashi [9]).

TRAIL-induced activation of NF-κB caused transcriptional upregulation of CX3CL1 in PANC-1 cancer cells [10]. CX3CL1 acted as a chemoattractant for CX3CR1 receptor–expressing cells. CX3CL1 enhanced the migration of PBMCs toward PANC-1 cancer cells. Knockdown of CX3CL1 in PANC-1 cells abolished TRAIL resistance inducing effects by PBMC co-culture, thus directly highlighting a paracrine role of CX3CL1 in apoptosis resistance in PDAC cells (Geismann [10]).

Receptor interacting protein (RIP) kinases crucially regulate crucial cell survival and death [11]. In apoptosis-sensitive cells, caspase-8–mediated cleavage of RIP1 resulted in rapid depletion of RIP1 and induction of apoptotic death. Whereas, in apoptosis-resistant cells, RIP1 was reported to be constitutively active and further activated NF-κB. Essentially, downregulation of caspase-8 by oncogenic miRNAs blocked RIP1 cleavage and induced NF-κB activation. NF-κB has been shown to transcriptionally upregulate wide variety of miRNAs which play instrumental role in metastasis (Jeon [11]).

Certain hints have emerged which also pinpoint to NF-κB–mediated upregulation of tumor suppressor miRNAs. For example, TRAIL-induced activation of NF-κB has also been shown to stimulate the expression of miR-146a [12]. It was shown that miR-146a directly targeted CXCR4 in MDA-MB-231 cancer cells. Intratumorally injected TRAIL caused marked reduction in the growth of the tumors derived from MDA-MB-231 cancer cells. Importantly, miR-146a was noted to be enhanced, but the levels of CXCR4 were found to be reduced in the tumor tissues of the mice intratumorally injected with TRAIL (Wang [12]).

NF-ҝB dualistically regulates TRAIL-mediated signaling. NF-ҝB transcriptionally enhanced the expression of antiapoptotic genes as well as prometastatic genes, but on a different node NF-ҝB is involved in the upregulation of tumor suppressor miRNAs as well. These findings are exciting and need additional research.

Concluding remarks

Seemingly, TRAIL-mediated signaling has been shown convincingly to inhibit carcinogenesis and metastasis, but still the prometastatic properties of TRAIL-driven pathway cannot be overlooked. We have summarized an intricate interplay between nonapoptotic signaling and cancer metastasis, hoping to provide reference information for more in-depth molecular research in this field in the future.

References

1. Hoogwater F.J, Nijkamp M.W, Smakman N, Steller E.J, Emmink B.L, Westendorp B.F, Raats D.A, Sprick M.R, Schaefer U, Van Houdt W.J, De Bruijn M.T, Schackmann R.C, Derksen P.W, Medema J.P, Walczak H, Borel Rinkes I.H, Kranenburg O. Oncogenic K-Ras turns death receptors into metastasis-promoting receptors in human and mouse colorectal cancer cells.  Gastroenterology . 2010;138(7):2357–2367. doi: 10.1053/j.gastro.2010.02.046.

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Chapter 2: Multiple roles of circulating tumor cells and exosomes in cancer metastasis

implications for therapeutic intervention

Chiara Martinelli     Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Pontedera, Italy

Abstract

Metastatic cancer is a prominent cause of death worldwide. Clinical evidences indicate that cancer patients affected by metastases display tumors diffused over the entire body that, once established, become very difficult to be eradicated and most of the times give a negative outcome. Both circulating tumor cells (CTCs) and exosomes can be detected in affected individuals' bodily fluids and participate to the metastatic cascade. Importantly, they are considered valuable predictors of therapeutic responses. In the last years, these biomaterials have been widely characterized, demonstrating their involvement in multiple steps of metastasis, and many strategies have been developed in order to isolate them. CTCs and exosomes have become fundamental tools for investigating and dissecting the cellular and molecular mechanisms responsible for the metastatic process, a quest everyday more critical for prevention and treatment of metastatic cancer.

Keywords

Biomarkers; Circulating tumor cells; Diagnosis; Exosomes; Metastasis; Therapy

List of abbreviations

AFM    Atomic force microscopy

AR-V7    Androgen receptor splice variant 7

ASC    Adipose-derived stem cell

CK    Cytokeratin

CTC    Circulating tumor cell

EBV    Epstein–Barr virus

EGFR    Epidermal growth factor receptor

ELISA    Enzyme-linked immunosorbent assay

EpCAM    Epithelial cell adhesion molecule

ER    Estrogen receptor

ESCRT    Endosomal sorting complexes required for transport

FACS    Fluorescence activated cell sorting

IGF1R    Insulin-like growth factor receptor 1

lncRNA    Long noncoding RNA

miRNA    microRNA

MNP    Magnetic nanoparticle

mRNA    Messenger RNA

MSC    Mesenchymal stem cell

NGS    Next-generation sequencing

NPC    Nasopharyngeal carcinoma

NSCLC    Non–small cell lung cancer

RBC    Red blood cell

RNA-seq    RNA sequencing

ROS    Reactive oxygen species

RT-PCR    Real-time polymerase chain reaction

shRNA    Short hairpin RNA

TEM    Transmission electron microscopy

WBC    White blood cell

μNMR    Micro-nuclear magnetic resonance

Introduction

Metastatic disease is currently considered the main cause of death for over 90% of cancer patients worldwide. Unraveling the cellular and molecular mechanisms and pathological features of metastasis is essential for managing its progression and, ideally, eradicating it. In the past, many difficulties have been associated with the investigation of metastatic lesions, mainly because of (1) their fast spreading in different organs and (2) the absence of reliable technologies to isolate circulating materials shed by aggressive solid tumors, such as circulating tumor cells (CTCs) and extracellular vesicles [1]. More recently, the development of devices allowing to separate and characterize at the molecular level single cells and exosomes has paved the way toward innovative diagnostic tools, able to improve prevention and treatment of metastatic cancer. However, one of the main difficulties related to diagnosis remains the fact that, even when a major metastasis becomes detectable, many other micrometastases might persist below the resolution limits of the diagnostic instruments, yet retarding the chance of efficiently targeting and blocking their progression [2]. Commonly, pathological features of metastases are deduced from the molecular characterization of primary tumor. Nevertheless, it has been demonstrated that it remains a molecularly distinct entity with respect to the CTCs able to settle in distant organs. Metastasis per se is a continuously evolving and heterogeneous disease [3].

Recently, the possibility to perform liquid biopsies isolating CTCs and exosomes has given the unique opportunity to obtain real-time and highly precise profiling of the metastatic process. Circulating materials carry a plethora of molecular information and, actually, they are considered effective biomarkers for tumor diagnosis, progression, and treatment monitoring. In particular, liquid biopsies based on microfluidics can be performed in patients affected by different types of cancer. It is necessary to consider that CTCs are very rare (less than 10 CTCs per million normal blood cells) [4]; however, they are an outstanding tool allowing to characterize living and proliferating cells detaching from the primary tumorigenic locus. Common devices applied for capturing CTCs recognize their cell surface markers and peculiar physical properties, as compared to normal blood cells