HOX Genes as Potential Markers of Circulating Tumour Cells.

Circulating tumour cells (CTCs) have significant diagnostic potential as they can reflect both the presence and recurrence of a wide range of cancers. However, this potential continues to be limited by the lack of robust and accessible isolation technologies. An alternative to isolation might be their direct detection amongst other peripheral blood cells, although this would require markers that allow them to be distinguished from an exceptionally high background signal. This review assesses the potential role of HOX genes, a family of homeodomain containing transcription factors with key roles in both embryonic development and oncogenesis, as unique and possibly disease specific markers of CTCs.

Resistance to HSP90 inhibition involving loss of MCL1 addiction.

Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.

RAN GTPase as a target for cancer therapy: Ran binding proteins.

The identification of a relevant effector of Ran GTPase (Ran) signaling and its pathways could provide a novel approach to cancer therapeutics. With recent research highlighting the significant relationship between Ran expression and the occurrence and progression of cancer, the development of a small molecule compound that would decrease the endogenous levels of Ran in the cell would have anti-mitotic effects and could lead to the development of new types of cancer therapeutics. In the absence of Ran binding proteins, Ran is expected to remain locked up in non-productive complexes with importins and is effectively removed from the system. Thus, Ran binding proteins present as a logical molecular target for the inhibition of Ran signaling within the cancer cell. Moreover, this family of proteins has been shown to have various other functions within the cell, some of which are also anti-neoplastic. The purpose of this review is to discuss Ran binding proteins and how their pathways may be exploited to provide an effective cancer treatment.

Wnt-ß-catenin-Tcf-4 signalling-modulated invasiveness is dependent on osteopontin expression in breast cancer.

BACKGROUND: We have previously demonstrated that Tcf-4 regulates osteopontin (OPN) in rat breast epithelial cells, Rama37. In this report, we have examined the importance of this regulation in human breast cancer. METHODS: The regulatory roles of Tcf-4 on cell invasion and OPN expression were investigated. The mRNA expression of Tcf-4 and OPN, and survival of breast cancer patients were correlated. RESULTS: Tcf-4 enhanced cell invasion in both MCF10AT and MDA MB 231 breast cancer cells by transcriptionally activating OPN expression. Osteopontin was activated by Wnt signalling in MDA MB 231 cells. Paradoxical results on Tcf-4-regulated OPN expression in MCF10AT (activation) and Rama37 (repression) cells were shown to be a result of differential Wnt signalling competency in MCF10AT and Rama37 cells. High levels of OPN and Tcf-4 mRNA expression were significantly associated with survival in breast cancer patients. Most importantly, Tcf-4-positive patients had a poorer prognosis when OPN was overexpressed, while OPN-negative patients had a better prognosis when Tcf-4 was overexpressed. CONCLUSION: Our results suggest that Tcf-4 can act as a repressor or activator of breast cancer progression by regulating OPN expression in a Wnt-dependent manner and that Tcf-4 and OPN together may be a novel prognostic indicator for breast cancer progression.

Expression of osteopontin coregulators in primary colorectal cancer and associated liver metastases.

BACKGROUND: A transcription regulatory complex (TRC) that includes Ets1, Ets2, PEA3 and ß-catenin/T-cell factors regulates osteopontin (OPN) that is implicated in colorectal cancer (CRC) dissemination. The consistency of OPN transcriptional control between primary CRC and metastases is unclear. This study investigates expression and prognostic significance of the OPN-TRC in primary human CRC and associated colorectal liver metastases (CRLM). METHODS: Osteopontin-TRC factors were assayed by digital microscopy in 38 primary CRCs and matched CRLM specimens and assessed against clinical prognosis. RESULTS: In primary CRC, OPN expression intensity correlated with that of its co-activators, PEA3 (r=0.600; P<0.01), Ets1 (r=0.552; P<0.01), Ets2 (r=0.521; P<0.01) and had prognostic significance. Osteopontin intensity in primary CRC inversely correlated with the interval between diagnosis and resection of CRLM. Overall OPN intensity was lower in CRLM than primary CRC and correlations with co-activators were weaker, for example, Ets1 (P=0.047), PEA3 (P=0.022) or nonsignificant (Ets2). The ratio of OPN expression in CRLM vs primary CRC had prognostic significance. CONCLUSION: This study supports transcriptional control of OPN by known coregulators in both primary and secondary CRC. Weaker associations in CRLM suggest involvement of other unknown factors possibly from the liver microenvironment or resulting from additional genetic or epigenetic changes that drive tumour metastatic capability in OPN transcriptional control.

Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression.

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system.

The yin and yang of vitamin D receptor (VDR) signaling in neoplastic progression: operational networks and tissue-specific growth control.

Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1alpha,25-(OH)2 D3 in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1alpha,25-(OH)2 D3. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1alpha,25-(OH)2 D3/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1alpha,25-(OH)2 D3 genomic and non-genomic actions, Ca2+ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.

Prostate cancer cells modulate osteoblast mineralisation and osteoclast differentiation through Id-1.

BACKGROUND: Id-1 is overexpressed in and correlated with metastatic potential of prostate cancer. The role of Id-1 in this metastatic process was further analysed. METHODS: Conditioned media from prostate cancer cells, expressing various levels of Id-1, were used to stimulate pre-osteoclast differentiation and osteoblast mineralisation. Downstream effectors of Id-1 were identified. Expressions of Id-1 and its downstream effectors in prostate cancers were studied using immunohistochemistry in a prostate cancer patient cohort (N=110). RESULTS: We found that conditioned media from LNCaP prostate cancer cells overexpressing Id-1 had a higher ability to drive osteoclast differentiation and a lower ability to stimulate osteoblast mineralisation than control, whereas conditioned media from PC3 prostate cancer cells with Id-1 knockdown were less able to stimulate osteoclast differentiation. Id-1 was found to negatively regulate TNF-beta and this correlation was confirmed in human prostate cancer specimens (P=0.03). Furthermore, addition of recombinant TNF-beta to LNCaP Id-1 cell-derived media blocked the effect of Id-1 overexpression on osteoblast mineralisation. CONCLUSION: In prostate cancer cells, the ability of Id-1 to modulate bone cell differentiation favouring metastatic bone disease is partially mediated by TNF-beta, and Id-1 could be a potential therapeutic target for prostate cancer to bone metastasis.

RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin.

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to alpha v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

Differential modulation of transcriptional activity of estrogen receptors by direct protein-protein interactions with the T cell factor family of transcription factors.

Two major signaling pathways, those triggered by estrogen (E(2)) and by the Wnt family, interact in the breast to cause growth and differentiation. The estrogen receptors ER(alpha) and ER(beta) are activated by binding E(2) and act as ligand-dependent transcription factors. The effector for the Wnt family is the Tcf family of transcription factors. Both sets of transcription factors recognize discrete but different nucleotide sequences in the promoters of their target genes. By using transient transfections of reporter constructs for the osteopontin and thymidine kinase promoters in rat mammary cells, we show that Tcf-4 antagonizes and Tcf-1 stimulates the effects of activated ER/E(2). For mutants of the former promoter, the stimulatory effects of ER(alpha)/E(2) can be made to be dependent on Tcf-1, and for the latter promoter the effects of the T cell factors (TCFs) are dependent on ER/E(2). Direct interaction between ERs and Tcfs either at the Tcf/ER(alpha)-binding site on the DNA or in the absence of DNA is established by gel retardation assays or by coimmunoprecipitation/biosensor methods, respectively. These results show that the two sets of transcription factors can interact directly, the interaction between ERs and Tcf-4 being antagonistic and that between ERs and Tcf-1 being synergistic on the activity of the promoters employed. Since Tcf-4 is the major Tcf family member in the breast, it is suggested that the antagonistic interaction is normally dominant in vivo in this tissue.

Metastasis-inducing dna regulates the expression of the osteopontin gene by binding the transcription factor Tcf-4.

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

Regulatory region of metastasis-inducing DNA is the binding site for T cell factor-4.

Small 1000 bp fragments of DNA derived from human malignant breast cancer cells have been isolated which, when transfected into a benign rat mammary cell line induce the production of osteopontin and thereby endow those cells with the capability to metastasize in syngeneic rats. Using transient transfections of an osteopontin promoter-reporter construct, we have now identified the active moiety in the metastasis-inducing DNA as the binding site for the T cell factor (Tcf) family of transcription factors and located Tcf-4, beta-catenin and E-cadherin in the relevant DNA complex in vitro. The regulatory effects of the metastasis-inducing DNAs are therefore exerted, at least in part, by a CAAAG sequence which can sequester Tcf-4, thereby promoting transcription of the direct effector for metastasis in this system, osteopontin.

Two separate mechanisms for ligand-independent activation of the estrogen receptor.

Transient transfection experiments in which three different estrogen response element-containing reporter genes were cotransfected into HeLa cells, together with constitutively expressed estrogen receptor (ER) constructs, demonstrate that activation of the transcription of the reporter genes by epidermal growth factor (EGF) and by cholera toxin with 3-isobutyl-1-methyl-xanthine, which elevate cellular cAMP, is dependent upon the presence of functional ER. Cotransfection of the reporter genes with truncated versions of the ER shows that the two non-ligand activators of ER require different regions of the receptor to produce their effects on transcription. EGF acts primarily by means of transactivation domain AF-1, whereas cAMP acts via transactivation domain AF-2 of the ER. A point mutation that removes a major site of inducible phosphorylation within the AF-1 domain of the ER abolishes the response to EGF, but the response to estradiol and cAMP is retained. Specific inhibition of cAMP-activated protein kinase (protein kinase A) prevents the response to elevated cAMP but does not affect EGF or estradiol responses. Overexpression of the protein kinase A catalytic subunit in HeLa cells results in an amplified response to estradiol, similar to that induced by cholera toxin with 3-isobutyl-1-methyl-xanthine. Comparable experiments performed using COS-1 cells produce similar results but also reveal cell type- and promoter-specific aspects of the activation mechanisms. Apparently, the ER may be activated by three different signal molecules, estradiol, EGF, and cAMP, each using a mechanism that is distinguishable from that of the others.

Interaction between estradiol and growth factors in the regulation of specific gene expression in MCF-7 human breast cancer cells.

The response of two endogenous, estrogen-induced genes, LIV-1 and pS2, to growth factor stimulation of MCF-7 cells was examined. Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin-like growth factor-1 (IGF-1) were each able to induce an increase in the two mRNAs in the absence of estradiol, and their effects were additive to that of an optimally inducing concentration (10(-8) M) of the hormone. Induction by EGF and TGF alpha, but not by IGF-1, were also additive to induction by a saturating concentration (2 microg/ml) of insulin. TGFbeta, an antimitogenic growth factor for MCF-7 cells, did not induce LIV-1 or pS2 mRNA but inhibited induction by estradiol. Increases in mRNA were shown to reflect increases in specific gene transcription. Induction by growth factors, but not by estradiol, was dependent upon protein synthesis. Induction by both growth factors and estradiol was inhibited by the pure antiestrogen, ICI 164384 (ICI), and by the mixed agonist/antagonist, tamoxifen. Despite differences in patterns of expression in vivo and in vitro, both LIV-1 and pS2 appeared to be responsive to growth factors via a mechanism distinct from that of estradiol but requiring the estrogen receptor.

Interaction between estradiol and cAMP in the regulation of specific gene expression.

The mRNA levels of LIV-1 and pS2, two estrogen-responsive genes, are increased by the agents, cholera toxin (CT) plus 3-isobutyl-l-methylxanthine (IBMX), which cause an increase in cAMP in MCF-7 human breast cancer cells. The simultaneous addition of estradiol and CT/IBMX results in a synergistic induction of the two mRNAs. The changes in mRNA reflect changes in transcription of the two genes. Interestingly, the addition of CT/IBMX to estradiol not only causes a greater increase in transcription rate but the increase is longer-lasting that seen with the hormone alone. Stimulation of mRNA levels by CT/IBMX, but not by estradiol, was prevented by cycloheximide. Stimulation by both estradiol and by CT/IBMX was prevented by the antiestrogen, ICI 164387. Transcription of LIV-1 and pS2 genes is by both estradiol and cAMP, via separate mechanisms both requiring the estrogen receptor.

Insulin/IGF-1 modulation of the expression of two estrogen-induced genes in MCF-7 cells.

Estrogen responses of human breast cancer cell lines have frequently been shown to be promoted by insulin. We have examined the action of insulin, and its interaction with estradiol, in regulating the expression of the estrogen-induced genes, LIV-1 and pS2. Both hormones cause increases in mRNA levels of the two genes but do so by distinct mechanisms. The concentration of insulin required to produce this effect suggests that it is acting via its ability to bind to the IGF-1 receptor. Both insulin and estradiol exert their effects at the level of transcription. Induction by insulin is dependent upon continued protein synthesis whereas induction by estradiol is not. Induction by both insulin and estradiol is prevented by the pure antiestrogen. ICI 164384, indicating the requirement for an activatable estrogen receptor. Insulin does not stimulate LIV-1 expression via the androgen receptor. These results demonstrate that both estradiol and insulin can stimulate the transcription of these estrogen-inducible genes, by separate mechanisms both of which involve the estrogen receptor.