Mesenchymal-epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer

As yet, there is no cure for metastatic breast cancer. Historically, considerable research effort has been concentrated on understanding the processes of metastasis, how a primary tumour locally invades and systemically disseminates using the phenotypic switching mechanism of epithelial to mesenchymal transition (EMT); however, much less is understood about how metastases are then formed. Breast cancer metastases often look (and may even function) as 'normal' breast tissue, a bizarre observation against the backdrop of the organ structure of the lung, liver, bone or brain. Mesenchymal to epithelial transition (MET), the opposite of EMT, has been proposed as a mechanism for establishment of the metastatic neoplasm, leading to questions such as: Can MET be clearly demonstrated in vivo? What factors cause this phenotypic switch within the cancer cell? Are these signals/factors derived from the metastatic site (soil) or expressed by the cancer cells themselves (seed)? How do the cancer cells then grow into a detectable secondary tumour and further disseminate? And finallyCan we design and develop therapies that may combat this dissemination switch? This review aims to address these important questions by evaluating long-standing paradigms and novel emerging concepts in the field of epithelial mesencyhmal plasticity.

Preclinical drug development must consider the impact on metastasis

Recently, two landmark reports on antiangiogenic therapy were published: Paez-Ribes and colleagues and Ebos and colleagues . The Board of the Metastasis Research Society (MRS) congratulates the authors for their informative articles that help to explain the puzzle of why antiangiogenic agents have had a relatively minor or no significant impact on patient survival. Using four model systems and several different strategies, these researchers showed that inhibition of angiogenesis reduced primary tumor growth and microvessel density in keeping with many earlier reports, but strikingly, accelerated invasion and metastasis.

lacZ transduced human breast cancer xenografts as an in vivo model for the study of invasion and metastasis

A number of human cancer cell lines have been described as being invasive and metastatic in immune incompetent animals. However, it is difficult to assess metastatic spread of a subcutaneously injected or inoculated cell line, since an exact detection of all microfoci of human tumour cells in the animals by usual histological procedures would require extensive sectioning of the whole animal. To overcome this problem, we transduced human breast cancer cells with a replication-defective Moloney murine leukaemia retroviral vector (M-MuLV) containing both neo(R) (neomycin resistance) and lacZ genes. The resulting cell lines were selected for antibiotic (G418) resistance, and cell-sorted for lacZ expression. lacZ continued to be expressed in cultured cells for at least 20 passages without further G418 selection. The lacE gene codes for β-D-galactosidase, and cells expressing this gene stain blue with the chromogenic substrate X-gal. The lacZ-expressing cells retained the pre-transduction ability to traverse Matrigel in vitro, to form subcutaneous tumours in nude mice, and to grow invasively with the formation of metastases. X-gal staining showed high specificity, staining the tumour cells but not the surrounding mouse tissue on either whole tissue blocks or histological sections. The staining procedure was highly sensitive, allowing detection of microfoci of human cancer cells, and quantitative estimation of the metastatic capacity of the cells. These results indicate that lacZ transduction of human tumour cells is a powerful means of studying human cancer cell invasion and metastases in vivo.

Mechanisms of tumour invasion and metastasis : emerging targets for therapy

The progression of a tumour from one of benign and delimited growth to one that is invasive and metastatic is the major cause of poor clinical outcome in cancer patients. The invasion and metastasis of tumours is a highly complex and multistep process that requires a tumour cell to modulate its ability to adhere, degrade the surrounding extracellular matrix, migrate, proliferate at a secondary site and stimulate angiogenesis. Knowledge of the process has greatly increased and this has resulted in the identification of a number of molecules that are fundamental to the process. The involvement of these molecules has been shown to relate not only to the survival and proliferation of the tumour cell but, also to the processes of tumour cell adhesion, migration, and the tumour cells ability to degrade and escape the primary site as well as play a role in angiogenesis. These molecules may provide important therapeutic targets that represent the ability to target specific steps in the process of invasion and metastasis and provide additional therapies. The review focuses on representative key targets in each of these processes and summarises the state of play in each case.

Assays for the study of human cancer cell invasion and metastasis

Two in vitro and two in vivo assays for the study of human cancer invasion and metastasis are described. The assays include in vitro invasiveness through an artificial basement membrane (Matrigel®), invasiveness and metastasis in nude mice of subcutaneously injected LacZ-transduced human tumor cells, in vitro adherence to basement membrane components, and LacZ-transduced human cancer cells injected intravenously into nude mice. In studies of the processes involved in human cancer cell invasion and metastasis, these four assays were found to be complementary, and thus provide a set of test systems for preclinical screening of agents which interfere with these processes.

Invasive and metastatic properties of MCF-7 cells and ras(H)-transfected MCF-7 cell lines

In vitro invasion and in vivo metastasis assays were performed with a panel of MCF-7 cells transfected with isogenic constructs of mutated ras(H) genes. Both increased levels of ras(H) expression and ras(H) oncogene activation increased activity of derivative cell lines in in vitro invasion assays. In vivo formation of spontaneous metastases was assessed after intradermal inoculation of MCF-7 cells in the vicinity of the mammary fat pads of ovariectomized nude mice. No metastases were seen in the absence of estradiol treatment of the mice. With estradiol supplementation of the mice both the ras(H)-transfected and control transfected cell lines gave a higher incidence of metastases than parental MCF-7 cells. Prolonged treatment of mice with exogenous estradiol (60 days vs. 21 days) resulted in more frequent metastases to liver and lung at the end of the 90-day observation period. In contrast to activated ras(H)-gene enhancement of metastatic capacity of rodent fibroblast and epithelial cell lines, there was no correlation of ras(H) expression with in vivo metastatic capacity of a human mammary carcinoma cell line.

BM18 : a novel androgen-dependent human prostate cancer xenograft model derived from a bone metastasis

BACKGROUND Androgen-dependent prostate cancer (PrCa) xenograft models are required to study PrCa biology in the clinically relevant in vivo environment. METHODS Human PrCa tissue from a femoral bone metastasis biopsy (BM18) was grown and passaged subcutaneously through male severe combined immune-deficient (SCID) mice. Human mitochondria (hMt), prostate specific antigen (PSA), androgen receptor (AR), cytokeratin-18 (CK-18), pan-cytokeratin, and high molecular weight-cytokeratin (HMW-CK) were assessed using immunohistochemistry (IHC). Surgical castration was performed to examine androgen dependence. Serum was collected pre- and post-castration for monitoring of PSA levels. RESULTS: BM18 stained positively for hMt, PSA, AR, CK-18, pan keratin, and negatively for HMW-CK, consistent with the staining observed in the original patient material. Androgen-deprivation induced tumor regression in 10/10 castrated male SCID mice. Serum PSA levels positively correlated with BM18 tumor size. CONCLUSIONS BM18 expresses PSA and AR, and rapidly regresses in response to androgen withdrawal. This provides a new clinically significant PrCa model for the study of androgen-dependent growth.

Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of b-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.

Rad51 supports triple negative breast cancer metastasis

In contrast to extensive studies on familial breast cancer, it is currently unclear whether defects in DNA double strand break (DSB) repair genes play a role in sporadic breast cancer development and progression. We performed analysis of immunohistochemistry in an independent cohort of 235 were sporadic breast tumours. This analysis suggested that RAD51 expression is increased during breast cancer progression and metastasis and an oncogenic role for RAD51 when deregulated. Subsequent knockdown of RAD51 repressed cancer cell migration in vitro and reduced primary tumor growth in a syngeneic mouse model in vivo. Loss of RAD51 also inhibited associated metastasis not only in syngeneic mice but human xenografts and changed the metastatic gene expression profile of cancer cells, consistent with inhibition of distant metastasis. This demonstrates for the first time a new function of RAD51 that may underlie the proclivity of patients with RAD51 overexpression to develop distant metastasis. RAD51 is a potential biomarker and attractive drug target for metastatic triple negative breast cancer, with the capability to extend the survival of patients, which is less than 6 months.

A tissue-engineered humanised xenograft model of human breast cancer-induced bone metastasis and bone colonisation

This thesis focuses on the development of a humanised mouse model to investigate human breast cancer metastasis to bone, an incurable disease presenting a major medical challenge in our society. The method is based on tissue-engineered constructs with human cells that generate a human bone-like organ within mice. This novel platform is further applied to mimic human-specific mechanisms of breast cancer metastasis and growth in human bone, and in particular the role of specific cell adhesion molecules in this process is closely investigated.

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy3-(p- nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

Comparison of GST theta activity in liver and kidney of four species

Glutathione transferases (GSTs) catalyzing the conjugation of glutathione with electrophilic substrates are important enzymes in the metabolism of xenobiotics. Several isozymes exhibit polymorphisms in humans. The two deletion polymorphisms of hGSTM1 and hGSTT1 result in total loss of enzyme activity in homozygous null genotype (GSTM1*0 and GSTT1*0 respectively) individuals (Seidegård et al. 1988; Pemble et al. 1994). Individuals that are heterozygous for hGSTT1 show distinctly lower enzyme activities than individuals carrying two functional alleles of hGSTT1 (Wiebel et al. 1996). A similar effect is conceivable for the hGSTM1 polymorphism but has not been verified so far.

Tetraspanins as regulators of the tumour microenvironment : implications for metastasis and therapeutic strategies

One of the hallmarks of cancer is the ability to activate invasion and metastasis (Hanahan et al., 2011). Cancer morbidity and mortality are largely related to the spread of the primary, localised tumour to adjacent and distant sites (Pantel et al., 2004). Appropriate management and treatment decisions of predicting metastatic disease at the time of diagnosis is thus crucial, which supports better understanding of the metastatic process. There are common events that occur during metastasis: dissociation from the primary tumour mass, reorganisation/remodelling of extracellular matrix, cell migration, recognition and transversal of endothelial cells and the vascular circulation and lodgement and proliferation within ectopic stroma (Wells, 2006). One of the key and initial events is the increased capability of cancer cells to move, escaping the regulation of normal physiological control. The cellular cytoskeleton plays an important role in cancer cell motility and active cytoskeletal rearrangement can result in metastatic disease. This active change in cytoskeletal dynamics results in manipulation of plasma membrane and cellular balance between cellular adhesion and motility which in turn determines cancer cell movement. Members of the tetraspanins play important roles in regulation of cancer migration and cancer-endothelial cell interactions, which are critical for cancer invasion and metastasis. Their involvements in active cytoskeletal dynamics, cancer metastasis and potential clinical application will be discussed in this review. In particular, tetraspanin member, CD151, is highlighted for its major role in cancer invasion and metastasis

Flupirtine enhances the anti-hyperalgesic effects of morphine in a rat model of prostate bone metastasis

Objective Current treatments for cancer pain are often inadequate, particularly when metastasis to bone is involved. The addition to the treatment regimen of another drug that has a complementary analgesic effect may increase the overall analgesia without the necessity to increase doses, thus avoiding dose-related side effects. This project investigated the synergistic effect of the addition of the potassium channel (KCNQ2–3) modulator flupirtine to morphine treatment in a rat model of prostate cancer-induced bone pain. Design Syngeneic prostate cancer cells were injected into the right tibia of male Wistar rats under anesthesia. This led to expanding tumor within the bone in 2 weeks, together with the concurrent development of hyperalgesia to noxious heat. Paw withdrawal thresholds from noxious heat were measured before and after the maximum non-sedating doses of morphine and flupirtine given alone and in combinations. Dose-response curves for morphine (0.13–5.0 mg/kg ip) and flupirtine (1.25–10.0 mg/kg ip) given alone and in fixed-dose combinations were plotted and subjected to an isobolographic analysis. Results Both morphine (ED50 = 0.74 mg/kg) and flupirtine (ED50 = 3.32 mg/kg) caused dose-related anti-hyperalgesia at doses that did not cause sedation. Isobolographic analysis revealed that there was a synergistic interaction between flupirtine and morphine. Addition of flupirtine to morphine treatment improved morphine anti-hyperalgesia, and resulted in the reversal of cancer-induced heat hyperalgesia. Conclusions These results suggest that flupirtine in combination with morphine may be useful clinically to provide better analgesia at lower morphine doses in the management of pain caused by tumors growing in bone.