MicroRNA-645 is an oncogenic regulator in colon cancer.

Despite advances in early diagnosis and the development of molecularly targeted therapy, curative treatment of colon cancer once it has metastasized is yet to be accomplished. This is closely associated with deregulated CRC cell proliferation and resistance to apoptosis. Here we reveal that upregulation of microRNA-645 (miR-645) through DNA copy number gain is responsible for enhanced proliferation and resistance to apoptosis in colon cancer. MiR-645 was upregulated in most colon cancer tissues related to adjacent normal mucosa. This appeared to be associated with amplification of a section of chromosome 20q13.13, where miR-645 is located. Inhibition of miR-645 reduced proliferation and enhanced sensitivity to apoptosis triggered by the chemotherapeutic drugs 5-fluorouracil and cisplatin in CRC cells, and retarded colon cancer xenograft growth. Conversely, overexpression of miR-645 in normal colon epithelial cells enhanced proliferation and triggered anchorage-independent cell growth. Although SRY-related HMG-box 30 (SOX30) was identified as a miR-645 target, its expression was only partially affected by miR-645, suggesting that miR-645 is a fine-tuning mechanism of SOX30 expression. Moreover, overexpression of SOX30 only moderately inhibited promotion of CRC cell proliferation by miR-645, indicating that miR-645 may have more targets that contribute to its pro-proliferation effect in colon cancer. Together, this study reveals that miR-645 can regulate oncogenesis in colon cancer with SOX30 being one of its targets.

INPP4B is an oncogenic regulator in human colon cancer.

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and is a tumor suppressor in some types of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Here we show that silencing of INPP4B blocks activation of Akt and serum- and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. Collectively, these results suggest that INPP4B may function as an oncogenic driver in colon cancer, with potential implications for targeting INPP4B as a novel approach to treat this disease.

MicroRNA-497 targets insulin-like growth factor 1 receptor and has a tumour suppressive role in human colorectal cancer.

Past studies have shown that amplified insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1-R) signalling has an important role in colorectal cancer (CRC) development, progression and resistance to treatment. In this report, we demonstrate that downregulation of microRNA-497 (miR-497) as a result of DNA copy number reduction is involved in upregulation of IGF1-R in CRC cells. MiR-497 and miR-195 of the miR-15/16/195/424/497 family that share the same 3' untranslated region (3'UTR) binding seed sequence and are predicted to target IGF1-R were concurrently downregulated in the majority of CRC tissues relative to paired adjacent normal mucosa. However, only overexpression of miR-497 led to suppression of the IGF1-R 3'UTR activity and downregulation of the endogenous IGF1-R protein in CRC cells. This was associated with inhibition of cell survival, proliferation and invasion, and increased sensitivity to apoptosis induced by various stimuli including the chemotherapeutic drugs cisplatin and 5-fluorouracil, and the death ligand tumour necrosis factor-related apoptosis-inducing ligand. The biological effect of miR-497 on CRC cells was largely mediated by inhibition of phosphatidylinositol 3-kinase/Akt signalling, as overexpression of an active form of Akt reversed its impact on cell survival and proliferation, recapitulating the effect of overexpression of IGF1-R. Downregulation of miR-497 and miR-195 appeared to associate with copy number loss of a segment of chromosome 17p13.1, where these miRs are located at proximity. Similarly to miR-195, the members of the same miR family, miR-424 that was upregulated, and miR-15a, miR-15b and miR-16 that were unaltered in expression in CRC tissues compared with paired adjacent normal mucosa, did not appear to have a role in regulating the expression of IGF1-R. Taken together, these results identify downregulation of miR-497 as an important mechanism of upregulation of IGF1-R in CRC cells that contributes to malignancy of CRC.

Suppression of PP2A is critical for protection of melanoma cells upon endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells, which involves dephosphorylation of Bim(EL) by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that ER stress reduces PP2A activity leading to increased ERK activation and subsequent phosphorylation and proteasomal degradation of Bim(EL). Despite sustained upregulation of Bim at the transcriptional level, the Bim(EL) protein expression was downregulated after an initial increase in melanoma cells subjected to pharmacological ER stress. This was mediated by increased activity of ERK, whereas the phosphatase activity of PP2A was reduced by ER stress in melanoma cells. The increase in ERK activation was, at least in part, due to reduced dephosphorylation by PP2A, which was associated with downregulation of the PP2A catalytic C subunit. Notably, instead of direct dephosphorylation of Bim(EL), PP2A inhibited its phosphorylation indirectly through dephosphorylation of ERK in melanoma cells. Taken together, these results identify downregualtion of PP2A activity as an important protective mechanism of melanoma cells against ER stress-induced apoptosis.

The Fat1 cadherin is overexpressed and an independent prognostic factor for survival in paired diagnosis-relapse samples of precursor B-cell acute lymphoblastic leukemia.

Improved survival of patients with acute lymphoblastic leukemia (ALL) has emerged from identifying new prognostic markers; however, 20% of children still suffer recurrence. Previously, the altered expression of Fat1 cadherin has been implicated in a number of solid tumors. In this report, in vitro analysis shows that Fat1 protein is expressed by a range of leukemia cell lines, but not by normal peripheral blood (PB) and bone marrow (BM) cells from healthy donors. In silico analysis of expression of array data from clinical leukemias found significant levels of Fat1 transcript in 11% of acute myeloid leukemia, 29% and 63% of ALL of B and T lineages, respectively, and little or no transcript present in normal PB or BM. Furthermore, in two independent studies of matched diagnosis-relapse of precursor B-cell (preB) ALL pediatric samples (n=32 and n=27), the level of Fat1 mRNA expression was prognostic at the time of diagnosis. High Fat1 mRNA expression was predictive of shorter relapse-free and overall survival, independent of other traditional prognostic markers, including white blood cell count, sex and age. The data presented demonstrate that Fat1 expression in preB-ALL has a role in the emergence of relapse and could provide a suitable therapeutic target in high-risk preB-ALL.

Ets-1 mediates upregulation of Mcl-1 downstream of XBP-1 in human melanoma cells upon ER stress.

Past studies have shown that upregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 is a major adaptive mechanism of melanoma cells to endoplasmic reticulum (ER) stress, and has an important role in resistance of the cells to apoptosis. In this study, we show that the increase in transcription of Mcl-1 in melanoma cells triggered by pharmacological ER stress inducers is mediated by the transcription factor Ets-1. By incremental deletion analysis of the Mcl-1 promoter, we identified a DNA fragment containing an Ets-1 binding site that is transcriptionally responsive to ER stress. Mutations in the Ets-1 binding site or knockdown of Ets-1 inhibited the increase in Mcl-1, indicating that Ets-1 has a critical role in transcriptional upregulation of Mcl-1. Similar to Mcl-1, Ets-1 was transcriptionally upregulated by ER stress. This was mediated by the IRE1α/XBP-1 branch of the unfolded protein response, as upregulation of Ets-1 was inhibited in melanoma cell lines deficient in IRE1α or XBP-1 established by short hairpin RNA knockdown. Activation of the PI3k/Akt pathway downstream of XBP-1 was also involved, in that inhibition of the pathway blocked upregulation of Ets-1. Inhibition of Ets-1 enhanced ER stress-induced apoptosis in melanoma cell lines and in fresh melanoma isolates, recapitulating the effect of inhibition of Mcl-1. These results reveal a key mechanism by which Mcl-1 is transcriptionally upregulated in melanoma cells by ER stress, and identify Ets-1 as a potential target for inhibition to sensitize melanoma cells to apoptosis.

Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIP(L) from degradation by the E3 ligase itch in human melanoma cells.

Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIP(L) as the levels of FLIP(L) were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIP(L), decreased the amount of the E3 ligase Itch associated with FLIP(L), and reduced FLIP(L) ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIP(L) and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIP(L) and thereby TRAIL-induced apoptosis in melanoma cells.

Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS.

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, Bim(S), remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF(V600E) triggers preferential splicing to produce Bim(S), which is particularly important in induction of apoptosis in B-RAF(V600E) melanoma cells. Although the specific B-RAF(V600E) inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim(EL), Bim(L), and Bim(S), at the protein and mRNA levels in B-RAF(V600E) melanoma cells, the increase in the ratios of Bim(S) mRNA to Bim(EL) and Bim(L) mRNA indicates that it favours Bim(S) splicing. Consistently, enforced expression of B-RAF(V600E) in wild-type B-RAF melanoma cells and melanocytes inhibits Bim(S) expression. The splicing factor SRp55 appears necessary for the increase in Bim(S) splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim(S) and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim(S) splicing is also mirrored in freshly isolated B-RAF(V600E) melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF(V600E) inhibitors.

Directional sensing of a phorbol ester gradient requires CD44 and is regulated by CD44 phosphorylation.

Cancer progression is associated with enhanced directional cell migration, both of the tumour cells invading into the stroma and stromal cells infiltrating the tumour site. In cell-based assays to study directional cell migration, phorbol esters are frequently used as a chemotactic agent. However, the molecular mechanism by which these activators of protein kinase C (PKC) result in the establishment of a polarized migratory phenotype is not known. Here we show that CD44 expression is essential for chemotaxis towards a phorbol ester gradient. In an investigation of CD44 phosphorylation kinetics in resting and stimulated cells, Ser316 was identified as a novel site of phosphorylation following activation of PKC. PKC does not phosphorylate Ser316 directly, but rather mediates the activation of downstream Ser316 kinase(s). In transfection studies, a phosphorylation-deficient Ser316 mutant was shown to act in a dominant-negative fashion to impair chemotaxis mediated by endogenous CD44 in response to a phorbol ester gradient. Importantly, this mutation had no effect on random cell motility or the ability of cells to migrate directionally towards a cocktail of chemoattractants. These studies demonstrate that CD44 functions to provide directional cues to migrating cells without affecting the motility apparatus.

The integrins alpha3beta1 and alpha6beta1 physically and functionally associate with CD36 in human melanoma cells. Requirement for the extracellular domain OF CD36.

Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins alpha(3)beta(1) and alpha(6)beta(1) and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of alpha(3) and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous beta(1) integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.

Engagement of variant CD44 confers resistance to anti-integrin antibody-mediated apoptosis in a colon carcinoma cell line.

The LIM 1863 colon carcinoma cell line grows as structured organoids around a central lumen, and we have previously demonstrated that the three-dimensional arrangement protects the individual cells from apoptosis induced by an anti-alpha v integrin antibody, 23C6 (Bates et al., 1994). Here we show that the intercellular forces which drive spheroid formation can be overcome by exposure of the cells to a collagen substrate, or more specifically through ligation of the CD44 receptor by a monoclonal antibody. Binding to immobilized anti-CD44 antibody induced a monolayer morphology which is accompanied by fibronectin production and secretion, and expression of the integrin alpha v beta 6. Significantly, the cells of the monolayer acquired resistance to 23C6 antibody-mediated apoptosis over time and this property was sustained even after removal from the monolayer. We provide data to show that this resistance is not dependent on monolayer morphology, constant engagement of the CD44 receptor, loss of the 23C6 antigen, or elevation of Bcl-2 or Bcl-XL protein. The CD44 expressed by LIM 1863 is shown to be the metastatic variant of the molecule therefore these results provide a possible explanation for the selective advantages conferred by expression of this variant for metastasizing colon cancer cells. Overall, the findings of this study support a model for the development of malignancy through the production of specific survival and growth signals as a direct consequence of a signaling event induced by stimulation of an epithelial variant of CD44.

Antibody binding to individual short consensus repeats of decay-accelerating factor enhances enterovirus cell attachment and infectivity.

Decay-accelerating factor (DAF), a widely expressed membrane complement-regulatory protein, is utilized as a cellular receptor by many human enteric pathogens. We show here that the binding of two enteroviruses to individual short consensus repeats (SCR) of DAF on the cell surface is greatly augmented by mAb binding to an alternate SCR: Coxsackievirus A21 binding to the SCR1 of DAF is increased by Ab binding to SCR3 and, conversely, Echovirus 7 binding to SCR3 is enhanced severalfold by Ab binding to SCR1. These Ab-induced increases in viral binding also resulted in increased viral infectivity. Using purified soluble DAF in a solid phase assay it was found that Ab binding to SCR1 is increased greatly in the presence of an Ab against SCR3 and, reciprocally, Ab against SCR1 greatly increases Ab binding to SCR3. In contrast to the results obtained with the larger viral particles, however, this reciprocal Ab-induced enhancement of binding is not seen when measuring Ab binding to membrane-bound DAF SCR on the cell surface. These findings provide a possible explanation for functional differences between membrane-bound and soluble DAF with implications for a potential role for DAF-binding molecules in regulating DAF function. This is the first demonstration of enhancement of viral infectivity mediated by Ab against the viral receptor.

CD36 forms covalently associated dimers and multimers in platelets and transfected COS-7 cells.

CD36 is a transmembrane glycoprotein expressed on the surface of a number of cell types. The analysis of CD36 from platelets using immunoblotting, gel filtration, and native PAGE indicated the presence of high molecular complexes exceeding the Mr of monomeric CD36. Experiments using transfected COS-7 cells revealed these complexes were homodimers and -multimers of CD36. The multimers could be dissociated by treatment with a reducing agent, indicating they were formed by intermolecular cysteine-bridging. Mutagenesis of the cDNA for CD36 implicated the cysteines in the extracellular domain of the molecule. The potential physiological roles of CD36 multimerisation are discussed.

Regulation of tumor cell motility and migration by CD63 in a human melanoma cell line.

CD63 belongs to the transmembrane 4 superfamily of membrane proteins and is expressed in several normal tissues as well as in melanoma cells. Previous reports have suggested that CD63 may play an important role in inhibiting melanoma progression, and this was supported by our studies showing that CD63 was associated with suppression of the growth of melanoma in nude mice. Recently, we and others have shown that CD63 may form noncovalent associations with beta1 integrins, which suggests that the function of CD63 may be related to that of integrins. To further explore the role of CD63 in melanoma, we transfected CD63 into a highly motile, CD63-negative melanoma cell line, KM3, which was shown to express alpha(v)beta5 as the predominant integrin with only trace amounts of beta1 integrins. Following transfection, CD63 was shown to associate with beta1 integrins, and beta1 expression appeared to be up-regulated. Cell motility in serum-containing media was markedly suppressed following transfection of CD63. This inhibition was potentiated by mAbs to CD63, and correlated with the level of CD63 expression. The CD63-transfected, but not the untransfected, melanoma cells showed increased adhesion and migration on the beta1 substrates, fibronectin, laminin, and collagen, whereas rates of migration were similar on the beta5 substrate, vitronectin. These results show that CD63 is involved in regulation of the motility of melanoma cells and their adhesion and migration on substrates associated with beta1 integrins. We suggest they provide further insights into the role of CD63 in tumor progression.

Stimulation of platelet activation and aggregation by a carboxyl-terminal peptide from thrombospondin binding to the integrin-associated protein receptor.

Thrombospondin, a major secretory product of the alpha-granules of activated platelets, is a large trimeric glycoprotein that plays an important role in platelet aggregation. On resting platelets, thrombospondin binds to a single receptor in a cation-independent manner, but upon platelet activation it binds at least two further, distinct receptors that are both dependent upon divalent cations. Each of these receptors on the platelet surface binds to different regions of the thrombospondin molecule, and such binding may be responsible for the multifunctional role of thrombospondin in aggregation. We show here that a peptide from the carboxyl terminus of thrombospondin, RFYVVMWK, directly and specifically induces the activation and aggregation of washed human platelets from different donors at concentrations of 5-25 microM. At lower concentrations the peptide synergizes with suboptimal concentrations of ADP to induce aggregation. Peptide affinity chromatography and immunoprecipitation with a monoclonal antibody were used to identify the receptor for the carboxyl-terminal peptide as the integrin-associated protein. The integrin-associated protein remained bound to the RFYVVMWK-containing peptide column when washed with a scrambled peptide in the presence of 5 mM EDTA, indicating a divalent cation-independent association. It is suggested that integrin-associated protein is the primary receptor for thrombospondin on the surface of resting platelets and is implicated in potentiating the platelet aggregation response.

CD63 associates with transmembrane 4 superfamily members, CD9 and CD81, and with beta 1 integrins in human melanoma.

CD63 belongs to the Transmembrane 4 superfamily (TM4SF) of membrane proteins whose functions are largely unknown. Previous results have suggested that CD63 may play an important role in the regulation of melanoma progression. To explore the role of CD63 in melanoma we have examined its association with other molecules by immunoprecipitation of CD63 from detergent induced lysates of melanoma cells. These results are the first to demonstrate an association between CD63 and two other TM4SF members, CD9 and CD81 in 2 human melanoma cell lines. We are also able to identify an association between CD9 and CD63 with beta 1 integrins in melanoma. The results suggest that CD63 is capable of forming multicomponent complexes with TM4SF members and beta 1 integrins on the surface of melanoma. These findings provide further insights into the function of CD63.

Effects of hormones and N6O2'-dibutyryl-adenosine 3' :5'-cyclic monophosphate, administered in vivo, on phosphate transport and metabolism in isolated rat liver mitochondria.

1. The administration of glucagon or N6O2'-dibutyryl cyclic AMP to fed rats by intraperitoneal injection was associated with a 2-fold increase in the amounts of endogenous Pi and ATP, and an increase in the rate and extent of transport of exogenous Pi (measured in either the presence or the absence of Ca2+) in mitochondria subsequently isolated from the liver. No change was observed in either the maximum rate of transport of exogenous Pi or in the rate of 32Pi exchange. 2. The changes induced by glucagon and dibutyryl cyclic AMP were markedly decreased by the co-administration of cycloheximide. 3. The administration of insulin to rats resulted in an increase of about 1.3-fold in the concentration of endogenous mitochondrial Pi 4. The amounts of endogenous Pi in mitochondrial isolated from the livers of starved rats were 3 times those in mitochondria isolated from fed animals. 5. It is concluded that the liver mitochondrial phosphatetransport system may be an important site of hormone action. 6. In the course of these experiments, it was shown that Ca2+ markedly stimulates mitochondrial phosphate transports.

The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumour cells.

Cyclic stimulation by Ca(2+) of respiration in mitochondria isolated from Ehrlich ascites-tumour cells occurs only when low phosphate concentrations (approx. 0.5mm) are also included in the incubation system. Under these circumstances the extra oxygen consumed is related stoicheiometrically to the amount of Ca(2+) taken up by the mitochondria; the values are similar to those obtained with mitochondria from rat liver in the absence of added phosphate. In contrast with liver mitochondria, up to 280nmol of Ca(2+)/mg of protein can be added to ascites mitochondria without causing any deleterious effect. Respiration in mitochondria isolated from the Yoshida ascites hepatoma (HA 130) and from the Morris hepatomas 5123C and 9618A is also stimulated by Ca(2+) in a cyclic manner. However, that in mitochondria from regenerating rat liver responds to Ca(2+) in the same way as those from normal rat liver. ADP-stimulated respiration in mitochondria from Ehrlich ascites tumour cells, but not from rat liver, is inhibited by low amounts of Ca(2+).