Pathobiology of cancer metastasis: a short account

ABSTRACT: Cancer-initiating cells display aberrant functional and phenotypic characteristics of normal stem cells from which they evolved by accumulation of multiple cytogenetic and/or epigenetic alterations. Signal transduction pathways which are essential for normal stem cell function are abnormally expressed by cancer cells, with a cancer cell phenotype playing an essential role in cancerization and metastasis.Local tumour progression, metastasis and metastatic tumour growth are mediated by direct cell-to-cell and paracrine reciprocal interactions between cancer cells and various stromal cells including fibroblasts, macrophages, bone marrow derived stem cells and progenitor cells. These interactions mediate breakdown of basement membrane barriers and angiogenesis both locally at the invasive front of the primary tumour and at the distant metastatic site; attract primary tumour cells to the candidate metastatic site; and promote proliferation, survival and growth of primary tumour cells and of metastatic cells at their distant site.It is the purpose of this article to highlight the analogies between some of the genetic programs of normal stem cells, and of cancer cells participating in the process of metastasis.     

The role of tumor microenvironment in prostate cancer bone metastasis

Prostate cancer (PCa) epithelial cells require a number of factors to facilitate their establishment and growth at a distant site of metastasis. Their ability to adapt to their microenvironment, proliferate and recruit an underlying stroma is integral to the survival and growth of the metastasis. PCa predominantly metastasizes to the bone, and bone metastases are the main cause of morbidity. The bone marrow provides a permissive environment for the formation of a metastasis. In some cases, the cells may remain dormant for some time, eventually proliferating in response to an unknown "trigger." The marrow is rich in progenitor cells that differentiate into numerous cell types, producing new blood vessels, supporting fibroblasts, and an underlying extracellular matrix (ECM) that form the reactive stroma. By secreting a number of cytokines, growth factors and proteases they recruit auxiliary cells required to produce a functional stroma. These components are involved in a reciprocal interaction between the stroma and the PCa cells, allowing for the growth and survival of the tumor. Left unchecked, once a PCa tumor has established itself in the bone marrow it will eventually replace the marrow, interrupting bone homeostasis and typically promoting an osteoblastic response in the bone including osteoclastic events. The abundant deposition of new woven bone results in nerve compression, bone pain and an increase in fractures in patients with PCa bone metastases. This review will examine the tumor microenvironment, its role in facilitating tumor dissemination, growth and the resultant pathologies associated with PCa bone metastasis.     

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.     

Activation of PPARγ in myeloid cells promotes lung cancer progression and metastasis

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) inhibits growth of cancer cells including non-small cell lung cancer (NSCLC). Clinically, use of thiazolidinediones, which are pharmacological activators of PPARγ is associated with a lower risk of developing lung cancer. However, the role of this pathway in lung cancer metastasis has not been examined well. The systemic effect of pioglitazone was examined in two models of lung cancer metastasis in immune-competent mice. In an orthotopic model, murine lung cancer cells implanted into the lungs of syngeneic mice metastasized to the liver and brain. As a second model, cancer cells injected subcutaneously metastasized to the lung. In both models systemic administration of pioglitazone increased the rate of metastasis. Examination of tissues from the orthotopic model demonstrated increased numbers of arginase I-positive macrophages in tumors from pioglitazone-treated animals. In co-culture experiments of cancer cells with bone marrow-derived macrophages, pioglitazone promoted arginase I expression in macrophages and this was dependent on the expression of PPARγ in the macrophages. To assess the contribution of PPARγ in macrophages to cancer progression, experiments were performed in bone marrow-transplanted animals receiving bone marrow from Lys-M-Cre+/PPARγ(flox/flox) mice, in which PPARγ is deleted specifically in myeloid cells (PPARγ-Mac(neg)), or control PPARγ(flox/flox) mice. In both models, mice receiving PPARγ-Mac(neg) bone marrow had a marked decrease in secondary tumors which was not significantly altered by treatment with pioglitazone. This was associated with decreased numbers of arginase I-positive cells in the lung. These data support a model in which activation of PPARγ may have opposing effects on tumor progression, with anti-tumorigenic effects on cancer cells, but pro-tumorigenic effects on cells of the microenvironment, specifically myeloid cells.     

Chemokines and cancer: migration, intracellular signalling and intercellular communication in the microenvironment

Inappropriate chemokine/receptor expression or regulation is linked to many diseases, especially those characterized by an excessive cellular infiltrate, such as rheumatoid arthritis and other inflammatory disorders. There is now overwhelming evidence that chemokines are also involved in the progression of cancer, where they function in several capacities. First, specific chemokine-receptor pairs are involved in tumour metastasis. This is not surprising, in view of their role as chemoattractants in cell migration. Secondly, chemokines help to shape the tumour microenvironment, often in favour of tumour growth and metastasis, by recruitment of leucocytes and activation of pro-inflammatory mediators. Emerging evidence suggests that chemokine receptor signalling also contributes to survival and proliferation, which may be particularly important for metastasized cells to adapt to foreign environments. However, there is considerable diversity and complexity in the chemokine network, both at the chemokine/receptor level and in the downstream signalling pathways they couple into, which may be key to a better understanding of how and why particular chemokines contribute to cancer growth and metastasis. Further investigation into these areas may identify targets that, if inhibited, could render cancer cells more susceptible to chemotherapy.     

Molecular pathway for cancer metastasis to bone

The molecular mechanism leading to the cancer metastasis to bone is poorly understood but yet determines prognosis and therapy. Here, we define a new molecular pathway that may account for the extraordinarily high osteotropism of prostate cancer. By using SPARC (secreted protein, acidic and rich in cysteine)-deficient mice and recombinant SPARC, we demonstrated that SPARC selectively supports the migration of highly metastatic relative to less metastatic prostate cancer cell lines to bone. Increased migration to SPARC can be traced to the activation of integrins alphaVbeta3 and alphaVbeta5 on tumor cells. Such activation is induced by an autocrine vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-2 loop on the tumor cells, which also supports the growth and proliferation of prostate cancer cells. A consequence of SPARC recognition by alphaVbeta5 is enhanced VEGF production. Thus, prostate cancer cells expressing VEGF/VEGFR-2 will activate alphaVbeta3 and alphaVbeta5 on their surface and use these integrins to migrate toward SPARC in bone. Within the bone environment, SPARC engagement of these integrins will stimulate growth of the tumor and further production of VEGF to support neoangiogenesis, thereby favoring the development of the metastatic tumor. Supporting this model, activated integrins were found to colocalize with VEGFR-2 in tissue samples of metastatic prostate tumors from patients.     

Role of copper in tumour angiogenesis--clinical implications.

The formation of new blood vessels is the initial step in progressive tumour development and metastasis. The first stage in tumour angiogenesis is the activation of endothelial cells. Copper ions stimulate proliferation and migration of endothelial cells. It has been shown that serum copper concentration increases as the cancer disease progresses and correlates with tumour incidence and burden. Copper ions also activate several proangiogenic factors, e.g., vascular endothelial growth factor, basic fibroblast growth factor, tumour necrosis factor alpha and interleukin 1. This review concerns a brief introduction into the basics of tumour blood vessel development as well as the regulatory mechanisms of this process. The role of copper ions in tumour angiogenesis is discussed. The new antiangiogenic therapies based on a reduction of copper levels in tumour microenvironment are reviewed.     

Folinic acid attenuates methotrexate chemotherapy-induced damages on bone growth mechanisms and pools of bone marrow stromal cells

Chemotherapy often induces bone growth defects in pediatric cancer patients; yet the underlying cellular mechanisms remain unclear and currently no preventative treatments are available. Using an acute chemotherapy model in young rats with the commonly used antimetabolite methotrexate (MTX), this study investigated damaging effects of five once-daily MTX injections and potential protective effects of supplementary treatment with antidote folinic acid (FA) on cellular activities in the tibial growth plate, metaphysis, and bone marrow. MTX suppressed proliferation and induced apoptosis of chondrocytes, and reduced collagen-II expression and growth plate thickness. It reduced production of primary spongiosa bone, volume of secondary spongiosa bone, and proliferation of metaphyseal osteoblasts, preosteoblasts and bone marrow stromal cells, with the cellular activities being most severely damaged on day 9 and returning to or towards near normal levels by day 14. On the other hand, proliferation of marrow pericytes was increased early after MTX treatment and during repair. FA supplementation significantly suppressed chondrocyte apoptosis, preserved chondrocyte proliferation and expression of collagen-II, and attenuated damaging effects on production of calcified cartilage and primary bone. The supplementation also significantly reduced MTX effects on proliferation of metaphyseal osteoblastic cells and of bone marrow stromal cells, and enhanced pericyte proliferation. These observations suggest that FA supplementation effectively attenuates MTX damage on cellular activities in producing calcified cartilage and primary trabecular bone and on pools of osteoblastic cells and marrow stromal cells, and that it enhances proliferation of mesenchymal progenitor cells during bone/bone marrow recovery.     

Pain control by ionizing radiation of bone metastasis

Pain treatment due to cancer is a large fraction of the care in a radiotherapy department. While radiation treatment is very effective in reducing pain, the pathophysiology of bone metastases remains very complex. Reducing the number of tumour cells by radiation will reduce the pressure in bone marrow, but the very rapid response to radiation treatment seen in some patients is probably related to the presence of highly radiosensitive inflammatory cells. In this review we give an overview of the pathophysiological mechanisms which lead to pain associated with bone metastasis and the impact of radiation treatment and other treatments on this mechanism.     

The role of the unfolded protein response in cancer progression: From oncogenesis to chemoresistance

Tumour cells endure both oncogenic and environmental stresses during cancer progression. Transformed cells must meet increased demands for protein and lipid production needed for rapid proliferation and must adapt to exist in an oxygen‐ and nutrient‐deprived environment. To overcome such challenges, cancer cells exploit intrinsic adaptive mechanisms such as the unfolded protein response (UPR). The UPR is a pro‐survival mechanism triggered by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), a condition referred to as ER stress. IRE1, PERK and ATF6 are three ER anchored transmembrane receptors. Upon induction of ER stress, they signal in a coordinated fashion to re‐establish ER homoeostasis, thus aiding cell survival. Over the past decade, evidence has emerged supporting a role for the UPR in the establishment and progression of several cancers, including breast cancer, prostate cancer and glioblastoma multiforme. This review discusses our current knowledge of the UPR during oncogenesis, tumour growth, metastasis and chemoresistance.     

SARI suppresses colitis‐associated cancer development by maintaining MCP‐1‐mediated tumour‐associated macrophage recruitment

SARI (suppressor of AP‐1, regulated by IFN) impaired tumour growth by promoting apoptosis and inhibiting cell proliferation and tumour angiogenesis in various cancers. However, the role of SARI in regulating tumour‐associated inflammation microenvironment is still elusive. In our study, the colitis‐dependent and ‐independent primary model were established in SARI deficiency mice and immuno‐reconstructive mice to investigate the functional role of SARI in regulating tumour‐associated inflammation microenvironment and primary colon cancer formation. The results have shown that SARI deficiency promotes colitis‐associated cancer (CAC) development only in the presence of colon inflammation. SARI inhibited tumour‐associated macrophages (TAM) infiltration in colon tissues, and SARI deficiency in bone marrow cells has no observed role in the promotion of intestinal tumorigenesis. Mechanism investigations indicated that SARI down‐regulates p‐STAT1 and STAT1 expression in colon cancer cells, following inhibition of MCP‐1/CCR2 axis activation during CAC development. Inverse correlations between SARI expression and macrophage infiltration, MCP‐1 expression and p‐STAT1 expression were also demonstrated in colon malignant tissues. Collectively, our results prove the inhibition role of SARI in colon cancer formation through regulating TAM infiltration.     

The role of the EGFR signaling in tumor microenvironment

The epidermal growth factor receptor (EGFR) family comprehends four different tyrosine kinases (EGFR, ErbB-2, ErbB-3, and ErbB-4) that are activated following binding to epidermal growth factor (EGF)-like growth factors. It has been long established that the EGFR system is involved in tumorigenesis. These proteins are frequently expressed in human carcinomas and support proliferation and survival of cancer cells. However, activation of the EGFR in non-malignant cell populations of the neoplastic microenvironment might also play an important role in cancer progression. EGFR signaling regulates in tumor cells the synthesis and secretion of several different angiogenic growth factors, including vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF). Overexpression of ErbB-2 also leads to increased expression of angiogenic growth factors, whereas treatment with anti-EGFR or anti-ErbB-2 agents produces a significant reduction of the synthesis of these proteins by cancer cells. EGFR expression and function in tumor-associated endothelial cells has also been described. Therefore, EGFR signaling might regulate angiogenesis both directly and indirectly. In addition, activation of EGFR is involved in the pathogenesis of bone metastases. Within the bone marrow microenvironment, cancer cells stimulate the synthesis of osteoclastogenic factors by residing stromal cells, a phenomenon that leads to bone destruction. It has been shown that EGFR signaling regulates the ability of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation. Taken together, these findings suggest that the EGFR system is an important mediator, within the tumor microenvironment, of autocrine and paracrine circuits that result in enhanced tumor growth.     

Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion,proliferation, migration and invasion of colon cancer cells

Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells.     

Bone morphogenetic proteins and their receptor signaling in prostate cancer

Bone morphogenetic proteins (BMPs) belong to the TGF-Beta superfamily and are vital bone inductive factors. BMPs also play important roles during embryonic development and the postnatal homeostasis of various organs and tissues, by controlling cellular differentiation, proliferation and apoptosis. Prostate cancer is the most common cancer in men in Western countries, with a high incidence of bone metastasis. Once bony metastasis developed, the condition is incurable, and contributes significant disease specific morbidity and mortality. However, the mechanisms underlying the development of bone metastasis remain unclear. BMPs have been implicated in the development of both primary and secondary tumors, particularly skeletal metastasis. Aberrations in BMPs signaling have also been identified in various neoplasms. Recently studies have also suggested a pivotal role in bone metastasis for Noggin, which is a BMP antagonist. In this review, we discuss the current knowledge of BMPs signaling, abnormalities which have been identified and their involvement in tumour progression, and particularly in the development of bone metastasis in prostate cancer.     

Integrin signalling during tumour progression

During progression from tumour growth to metastasis, specific integrin signals enable cancer cells to detach from neighbouring cells, re-orientate their polarity during migration, and survive and proliferate in foreign microenvironments. There is increasing evidence that certain integrins associate with receptor tyrosine kinases (RTKs) to activate signalling pathways that are necessary for tumour invasion and metastasis. The effect of these integrins might be especially important in cancer cells that have activating mutations, or amplifications, of the genes that encode these RTKs.