Targeting KRas-dependent tumour growth, circulating tumour cells and metastasis in vivo by clinically significant miR-193a-3p.

KRas is mutated in a significant number of human cancers and so there is an urgent therapeutic need to target KRas signalling. To target KRas in lung cancers we used a systems approach of integrating a genome-wide miRNA screen with patient-derived phospho-proteomic signatures of the KRas downstream pathway, and identified miR-193a-3p, which directly targets KRas. Unique aspects of miR-193a-3p biology include two functionally independent target sites in the KRas 3'UTR and clinically significant correlation between miR-193a-3p and KRas expression in patients. Rescue experiments with mutated KRas 3'UTR showed very significantly that the anti-tumour effect of miR-193a-3p is via specific direct targeting of KRas and not due to other targets. Ex vivo and in vivo studies utilizing nanoliposome packaged miR-193a-3p demonstrated significant inhibition of tumour growth, circulating tumour cell viability and decreased metastasis. These studies show the broader applicability of using miR-193a-3p as a therapeutic agent to target KRas-mutant cancer.

Functional proteomics identifies miRNAs to target a p27/Myc/phospho-Rb signature in breast and ovarian cancer.

The myc oncogene is overexpressed in almost half of all breast and ovarian cancers, but attempts at therapeutic interventions against myc have proven to be challenging. Myc regulates multiple biological processes, including the cell cycle, and as such is associated with cell proliferation and tumor progression. We identified a protein signature of high myc, low p27 and high phospho-Rb significantly correlated with poor patient survival in breast and ovarian cancers. Screening of a miRNA library by functional proteomics in multiple cell lines and integration of data from patient tumors revealed a panel of five microRNAs (miRNAs) (miR-124, miR-365, miR-34b*, miR-18a and miR-506) as potential tumor suppressors capable of reversing the p27/myc/phospho-Rb protein signature. Mechanistic studies revealed an RNA-activation function of miR-124 resulting in direct induction of p27 protein levels by binding to and inducing transcription on the p27 promoter region leading to a subsequent G1 arrest. Additionally, in vivo studies utilizing a xenograft model demonstrated that nanoparticle-mediated delivery of miR-124 could reduce tumor growth and sensitize cells to etoposide, suggesting a clinical application of miRNAs as therapeutics to target the functional effect of myc on tumor growth.

Kinome siRNA-phosphoproteomic screen identifies networks regulating AKT signaling.

To identify regulators of intracellular signaling, we targeted 541 kinases and kinase-related molecules with small interfering RNAs (siRNAs), and determined their effects on signaling with a functional proteomics reverse-phase protein array (RPPA) platform assessing 42 phospho and total proteins. The kinome-wide screen demonstrated a strong inverse correlation between phosphorylation of AKT and mitogen-activated protein kinase (MAPK) with 115 genes that, when targeted by siRNAs, demonstrated opposite effects on MAPK and AKT phosphorylation. Network-based analysis identified the MAPK subnetwork of genes along with p70S6K and FRAP1 as the most prominent targets that increased phosphorylation of AKT, a key regulator of cell survival. The regulatory loops induced by the MAPK pathway are dependent on tuberous sclerosis complex 2 but demonstrate a lesser dependence on p70S6K than the previously identified FRAP1 feedback loop. The siRNA screen also revealed novel bi-directionality in the AKT and GSK3 (Glycogen synthase kinase 3) interaction, whereby genetic ablation of GSK3 significantly blocks AKT phosphorylation, an unexpected observation as GSK3 has only been predicted to be downstream of AKT. This method uncovered novel modulators of AKT phosphorylation and facilitated the mapping of regulatory loops.

Involvement of the mt1 melatonin receptor in human breast cancer.

Two putative melatonin receptors have been described including the cell surface G-protein-linked receptors, mt1 and MT2, and the nuclear retinoic orphan receptor alpha (RORalpha). The mt1 receptor, but not the MT2 receptor, is expressed in human breast tumor cell lines, and melatonin-induced growth suppression can be mimicked by the mt1 and MT2 agonist, AMMTC, and blocked by the antagonist, CBPT. RORalpha receptors are also expressed in MCF-7 breast cancer cells and the putative RORalpha agonist CPG-52608 inhibits MCF-7 cell growth but with a very different dose-response than melatonin. Finally, melatonin and AMMTC, but not CPG-52608, can repress RORalpha transcriptional activity in MCF-7 cells.

Melatonin inhibits estrogen receptor transactivation and cAMP levels in breast cancer cells.

We have previously demonstrated that the pineal hormone, melatonin, can inhibit the growth of estrogen receptor-alpha (ERalpha)-positive breast cancer cells and suppress ERalpha gene transcription. To investigate the relationship between the estrogen response pathway and melatonin's growth inhibition, ERalpha-positive MCF-7 human breast cancer cells were transiently transfected with an estrogen response element (ERE) luciferase reporter construct and then treated with melatonin (10(-9)-10(-6) M) for 30 min followed by 10(-9) M 17-beta-estradiol (E2) or treated with each compound alone. Melatonin pre-treatment significantly reduced E2-induced ERalpha transactivation and ERalpha-ERE binding activity. We also conducted experiments to determine if melatonin modulates cAMP levels in MCF-7 cells. Melatonin inhibited the forskolin-induced and E2-induced elevation of cAMP levels by 57 and 45%, respectively. These data indicate that melatonin can act as a biological modifier to affect ERalpha transcriptional activity by regulating signal transduction pathways which impinge on the ERalpha and by altering E2-mediated ERalpha transactivation and ERalpha DNA binding activity.

Transcriptional repression of RORalpha activity in human breast cancer cells by melatonin.

Melatonin has repeatedly been shown to inhibit the proliferation of MCF-7 human breast cancer cells. Previous reports suggest that the actions of melatonin can be mediated either through G-protein coupled membrane receptors or via retinoid orphan receptors (RORalphas). In this study, we demonstrated the expression of RORalpha2, 3, and 4 transcripts in MCF-7 cells. These cells exhibited a high basal level of RORalpha transcriptional activity, which was further stimulated by serum. In the presence of serum, RORalpha transactivation and DNA-binding activity was repressed by melatonin even though melatonin had no effect on RORalpha protein levels. We found that RORalpha transcriptional activity in MCF-7 cells was regulated by modulators of the Ca2+/CaM signaling pathway. Given that melatonin has been reported to modulate the Ca2+/CaM signaling pathway in other tissues, our data indicate that melatonin may affect RORalpha transcriptional activity, expression of RORalpha regulated genes, and even breast cancer cell proliferation via modulation of the Ca2+/CaM signaling pathway.

G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation.

Extracellular signals when routed through signaling pathways that use heterotrimeric G proteins can engage multiple signaling pathways leading to diverse biological consequences. One locus at which signal sorting occurs is at the level of G proteins. G protein alpha-subunits appear to be capable of interacting with different effectors leading to engagement of distinct signaling pathways. Regulation of different pathways in turn leads to different biological outcomes. The process of neoplastic transformation is controlled to a large extent through the activation and inhibition of signaling pathways. Signaling pathways such as the Ras-MAPK, v-Src-Stat3 pathways are activated in the process of transformation. Expression of activated Galpha subunits have been shown to cause transformation of cells. While activation of the MAPK 1,2 pathway by various Galpha subunits has been reported for several years, recent studies show the activation and involvement of Src and Stat3 pathways in Galphao and Galphai mediated transformation of cells. Recent studies also suggest that both Galphai and Galphas may be able to interact with and activate Src. The activation of Src and Stat3 by G proteins has also been demonstrated by ligand-induced activation of G protein receptors. So increasingly it is becoming clear that the Src and Stat3 pathways are potential effectors for G proteins and that they may play a role in G protein function.

Differential responsiveness of MCF-7 human breast cancer cell line stocks to the pineal hormone, melatonin.

The estrogen receptor (ER)-positive MCF-7 human breast cancer cell line has been used extensively for the study of estrogen-responsive human breast cancer. However, various levels of estrogen responsiveness have been described in different stocks of MCF-7 cells. Because we have previously shown that the pineal hormone, melatonin, inhibits proliferation of MCF-7 cells and can modulate ER expression and transactivation, we investigated if various stocks of MCF-7 cells exhibit a differential responsiveness to the anti-proliferative effects of melatonin and the possible mechanisms involved. The MCF-7 stocks (M, O, H) were examined for: (1) mitogenic response to estradiol; (2) steady-state ER mRNA levels; (3) expression of the mt1 melatonin membrane receptor; (4) growth inhibition by melatonin; and (5) melatonin's modulation of expression of the ER and the estrogen-regulated genes, PgR, TGFbeta and pS2. For all of these parameters, there was a stock-specific response which showed: MCF-7M > MCF-7O > MCF-7H. These results demonstrate that there are significant differences in the responsiveness of various stocks of MCF-7 breast cancer cells to the growth-inhibitory effects of melatonin which can be correlated with both the level of ER mRNA expression and the degree of estrogen-responsiveness. These findings suggest that not only may these differences have some impact on the cells' estrogen-response pathway, but also that the primary growth-inhibitory effects of melatonin are transduced through the membrane-associated G-protein coupled mt1 melatonin receptor.

Stat3-mediated transformation of NIH-3T3 cells by the constitutively active Q205L Galphao protein.

Expression of Q205L Galphao (Galphao*), an alpha subunit of heterotrimeric guanine nucleotide-binding proteins (G proteins) that lacks guanosine triphosphatase (GTPase) activity in NIH-3T3 cells, results in transformation. Expression of Galphao* in NIH-3T3 cells activated signal transducer and activator of transcription 3 (Stat3) but not mitogen-activated protein (MAP) kinases 1 or 2. Coexpression of dominant negative Stat3 inhibited Galphao*-induced transformation of NIH-3T3 cells and activation of endogenous Stat3. Furthermore, Galphao* expression increased activity of the tyrosine kinase c-Src, and the Galphao*-induced activation of Stat3 was blocked by expression of Csk (carboxyl-terminal Src kinase), which inactivates c-Src. The results indicate that Stat3 can function as a downstream effector for Galphao* and mediate its biological effects.

Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors.

The pineal hormone, melatonin, inhibits proliferation of estrogen receptor (ER)-positive MCF-7 human breast cancer cells, modulates both ER mRNA and protein expression, and appears to be serum dependent, indicating interaction between melatonin and serum components. To examine the effects of melatonin on ER activity, ER transactivation assays were performed by transiently transfecting MCF-7 cells with an ERE-luciferase reporter construct. MCF-7 cells pre-treated with melatonin for as little as 5 min followed by either epidermal growth factor (EGF) or insulin resulted in the estrogen-independent transactivation of the ER. None of the compounds when used alone transactivated the ER. The ability of melatonin and EGF to transactivate the ER was abolished by the addition of the antiestrogen, ICI 164384, suggesting that melatonin and EGF co-operate to transactivate the ER. The modulation of ER transactivation was associated with changes in mitogen activated protein kinase activity and ER phosphorylation. This ER transactivation was blocked by pertussis toxin, a Galpha i-protein-coupled receptor inhibitor, suggesting cross talk between the G-protein-coupled melatonin receptor pathway and the EGF/insulin tyrosine kinase receptor pathways in modulating ER transactivation. Exactly how the ability of melatonin in combination with EGF to transactivate the ER relates to melatonin's observed growth suppressive effects is not clear. It is possible that, although melatonin and EGF transactivate the ER, this transactivation does not result in the full transcription of estrogen-responsive genes, but rather, makes the ER refractory to activation by estradiol, thus, blocking the mitogenic actions of estradiol.

A sequential treatment regimen with melatonin and all-trans retinoic acid induces apoptosis in MCF-7 tumour cells.

Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer.

Expression of Q227L-galphas in MCF-7 human breast cancer cells inhibits tumorigenesis.

The effects of expression of mutant (Q227L)-activated Galphas and elevation of cAMP on mitogen-activating protein kinase (MAPK) activity and the transformed phenotype were studied in the MCF-7 human mammary epithelial cell line. Elevation of cAMP partially inhibited the epidermal growth factor-stimulated DNA synthesis and the intrinsic MAPK (ERK-1 and ERK-2) of serum-starved MCF-7 cells. Addition of 8Br-cAMP or expression of mutant (Q227L)-activated Galphas in MCF-7 cells blocked the ability of these cells to grow in an anchorage-independent manner, as assessed by colony formation in soft agar. 8Br-cAMP in the culture medium also blocked estrogen stimulation of MCF-7 cell proliferation in vitro. MCF-7 cells expressing Q227L-Galphas grew very slowly in vitro, and when these cells were injected s.c. into athymic mice implanted with estrogen pellets, the frequency of tumor formation was reduced greatly and the sizes of the tumors formed were much smaller than those in mice injected with MCF-7 cells that had been transfected with the empty vector. These results indicate that the intracellular levels of cAMP in transformed mammary epithelial cells can be a crucial factor in determining the expression of the transformed phenotype. Interactions between the Gs/adenylyl cyclase and MAPK-1,2 signaling pathways could be one mechanism by which expression of the transformed phenotype in mammary epithelial cells are regulated.

Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box-binding protein, TBP.

We previously demonstrated that an Sp1-dependent reporter gene is preferentially expressed in G2 of the 1-cell mouse embryo following microinjection of the male pronucleus when compared to microinjection of the female pronucleus (P.T. Ram and R.M. Schultz, 1993, Dev. Biol. 156, 552-556). We also noted that expression of the reporter gene is not observed following microinjection of the germinal vesicle of the fully grown oocyte. In the present study, we examined expression of this reporter gene during oocyte growth, as well as the nuclear concentration of two transcription factors, Sp1 and the TATA box-binding protein, TBP, during oocyte growth and the first cell cycle. The extent of reporter gene expression decreases during oocyte growth and this decrease correlates with the decrease in nuclear concentration of Sp1, as determined by confocal immunofluorescent microscopy. In addition, results of immunoblotting experiments also indicate a similar decrease in the total concentration of Sp1 during oocyte growth. The nuclear concentration of TBP also decreases during oocyte growth, as determined by confocal immunofluorescent microscopy. Following fertilization, the pronuclear concentration of these two transcription factors increases in a time-dependent fashion and the concentration of each is greater in the male pronucleus as compared to the female pronucleus. For each pronucleus and for each transcription factor, this increase in nuclear concentration is inhibited by aphidicolin, which inhibits DNA synthesis. Last, the increase in nuclear concentration of these two proteins observed between the 1-cell and 2-cell stages does not require transcription or cytokinesis.

Expression patterns of novel genes during mouse preimplantation embryogenesis.

Little is known about the repertoire of genes expressed following zygotic gene activation, which occurs during the two-cell stage in the mouse. As an initial attempt to isolate novel genes, we used previously prepared two-cell and two-cell subtraction cDNA libraries (Rothstein et al., Genes Dev 6:1190-1201, 1992) to isolate a panel of seven cDNA clones. Three cDNAs had no match in the current DNA sequence data banks and three others revealed sequence homology to portions of sequences in the data banks. One cDNA was 90% homologous to the ras-related gene Krev/rap 1A. The temporal patterns of expression of these genes during oocyte maturation and preimplantation development were analyzed by a reverse transcription-polymerase chain reaction (RT-PCR) assay developed to measure relative levels of mRNAs. Three distinct temporal patterns of expression, designated Classes 1-3, were found. The two Class 1 genes displayed an actin-like pattern, with a gradual decline in expression during oocyte maturation and through the two-cell stage, followed by increases at the eight-cell and/or blastocyst stages. The four genes in Class 2 were expressed at relatively high levels during oocyte maturation and through the one-cell stage and then declined abruptly between the one- and two-cell stages; an increase then occurred at the eight-cell and/or blastocyst stages. The expression of the gene in Class 3 declined during oocyte maturation, but then showed a transient increase at the one-cell stage, with only a very slight increase in synthesis at either the eight-cell or blastocyst stage.

Reporter gene expression in G2 of the 1-cell mouse embryo.

Similar amounts of expression of a luciferase reporter gene that is driven by the SV40 early promoter are detected in G2 of 1-cell mouse embryo following injection of the male pronucleus in either early or mid/late S. No luciferase activity is observed when the male pronucleus is injected in early S and the embryos analyzed for expression in late S. Last, no luciferase activity is observed when the female pronucleus is injected in mid/late S and the embryos analyzed for luciferase activity in G2 of the first cell cycle. These results suggest that zygotic gene activation initiates in the 1-cell embryo rather than in the 2-cell embryo, as previously thought, and that differences between the transcriptional activity of male and female pronuclei exist.