Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors.

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.

Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis.

Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.

Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing.

Homology PCR has been used to identify receptor tyrosine kinases (RTKs) expressed during activation of rat hepatic stellate cells, the key fibrogenic mesenchymal element in the liver. Partial cDNAs encoding several RTKs were cloned from stellate cells activated in vivo, including those of Flt-1, Flk-1, c-met, PDGFR, and Tyro10/DDR2. RNAse protection from cells activated in vivo demonstrated biphasic induction of flt-1 and flk-1 mRNAs, receptors for vascular endothelial growth factor (VEGF). Culture-activation of stellate cells was associated with increased [125I]VEGF binding and Flt-1 and Flk-1 receptor protein. Induction of VEGF binding sites correlated with an 2.5-fold increase in DNA synthesis in response to VEGF, but only if cells were activated by growth on collagen 1, whereas cells maintained in a quiescent state on a basement membrane-like substratum (EHS matrix) were nonproliferative. In both stellate and endothelial cells VEGF-induced mitogenesis was augmented by co-incubation with basic fibroblast growth factor (bFGF), a cytokine with known synergy with VEGF. These findings suggest that the cellular targets of VEGF in liver may not be confined to sinusoidal endothelial cells, and that VEGF responses reflect combined effects on both hepatic stellate cells and sinusoidal endothelium.

Induction of vascular endothelial growth factor by insulin-like growth factor 1 in colorectal carcinoma.

Vascular endothelial growth factor (VEGF) is an angiogenic hormone that is produced by and supports the growth of many types of malignancies. The present study shows that insulin-like growth factor 1 (IGF-I), a mitogen that promotes the propagation of cancers through autocrine and paracrine mechanisms, increases the expression of mRNA for VEGF and production of VEGF protein by COLO 205 colon carcinoma cells. IGF-I also induces expression of VEGF mRNA in SW620, LSLiM6, and HCT15 colon carcinoma cells showing that this is a common response to IGF-I. Whereas IGF-I induced VEGF mRNA in each cell line examined (2.3-12-fold), it induced proliferation only in COLO 205 and LSLiM6 cells. Thus, the proliferative response induced by IGF-I and its ability to induce VEGF occur through distinguishable mechanisms. IGF-I increases the cellular content of VEGF mRNA by increasing the rate of transcription (5-fold after 4 h) and also by increasing the half-life of VEGF mRNA (0.6 +/- 0.07 h in control cells to 2.0 +/- 0.37 h in IGF-I-treated cells). Monoclonal antibody (alphaIR3) directed against the type 1 IGF receptor significantly attenuated the ability of IGF-I to promote expression of VEGF mRNA. Interestingly, by itself alphaIR3 acted as a weak agonist and induced a modest increase in the cellular content of VEGF mRNA. alphaIR3 also promoted tyrosine phosphorylation of the beta subunit of the IGF-I receptor, and the magnitude of this response was comparable with that induced by IGF-I. These observations point to a nonlinear relationship between activation of the IGF-I receptor and induction of VEGF mRNA. Thus, in addition to its direct, growth stimulatory effect on transformed cells, IGF-I induces the expression of VEGF which can promote the progression of cancer by regulating the development of new blood vessels.

Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis.

To investigate the relationship between angiogenesis and hepatic tumorigenesis, we examined the expression of vascular endothelial growth factor (VEGF) in 8 human colon carcinoma cell lines and in 30 human colorectal cancer liver metastases. Abundant message for VEGF was found in all tumors, localized to the malignant cells within each neoplasm. Two receptors for VEGF, KDR and flt1, were also demonstrated in most of the tumors examined. KDR and flt1 mRNA were limited to tumor endothelial cells and were more strongly expressed in the hepatic metastases than in the sinusoidal endothelium of the surrounding liver parenchyma. VEGF monoclonal antibody administration in tumor-bearing athymic mice led to a dose- and time-dependent inhibition of growth of subcutaneous xenografts and to a marked reduction in the number and size of experimental liver metastases. In hepatic metastases of VEGF antibody-treated mice, neither blood vessels nor expression of the mouse KDR homologue flk-1 could be demonstrated. These data indicate that VEGF is a commonly expressed angiogenic factor in human colorectal cancer metastases, that VEGF receptors are up-regulated as a concomitant of hepatic tumorigenesis, and that modulation of VEGF gene expression or activity may represent a potentially effective antineoplastic therapy in colorectal cancer.

Recombinant IL-4 inhibits IL-6 synthesis by adherent peripheral blood cells in vitro.

IL-4 has been shown to participate in interregulatory networks with other cytokines including IL-2, IFN-gamma, IL-1 beta, and TNF-alpha. The ability of recombinant IL-4 (rIL-4) to modulate the synthesis and release of IL-6 was investigated in vitro. LPS-stimulated adherent cells (AC) from human peripheral blood secreted IL-6 as determined by ELISA and bioassay with the B9 hybridoma cell line. The secretion of IL-6 peaked between 12-16 h after LPS stimulation. The addition of rIL-4 inhibited LPS-induced IL-6 production measured at 8 h. The inhibitory effect of rIL-4 on IL-6 secretion was dose-dependent and occurred at doses as low as 0.001 U/ml (0.01 pg/ml). Recombinant IL-4 was added to the cultures for various periods and removed by aspiration of the medium and washing the cells. The subsequent LPS-induction of IL-6 secretion was reduced in cultures exposed to rIL-4 for as little as 5 minutes indicating that the effect of rIL-4 on the monocytes was rapid and irreversible. Northern blot analysis revealed that the effect of rIL4 on LPS-induced production of IL-6 by AC occurred at least in part at the level of transcription. In contrast, PMA-induced IL-6 gene transcription was not affected by rIL-4. These findings indicate that rIL-4 can regulate the synthesis of IL-6 by adherent peripheral blood mononuclear cells.

Use of recombinant interferon in HSV-1 recurrence in the rabbit.

The use of recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing shedding of herpes simplex virus type-1 induced by iontophoresis of 6-hydroxydopamine and epinephrine. A post stimulation treatment schedule of RIFN alpha D, one drop four times a day was as effective as pretreatment, using the same dose regimen. The levels of interferon (IFN) present in the assay system are not sufficient to produce an antiviral effect. The levels of IFN required to suppress cell growth are substantially higher than the concentrations detected in the assay system.

Interferon effects on herpes simplex virus in rabbit and human cell cultures.

The effects of four subtypes of recombinant human alpha interferon (RIFN alpha), (A,B,D, and the hybrid A/D) were tested on six strains of herpes simplex virus (HSV). RIFN alpha -D was the most effective subtype in rabbit kidney cells, which is consistent with our previous in vivo results in the rabbit herpetic keratitis model. In human corneal cells, however, RIFN alpha -D was one of the least effective IFN subtypes tested. Conversely, RIFN alpha-A appeared to be relatively more effective in the human corneal cells than in the rabbit kidney cells, but RIFN alpha -B and RIFN alpha -A/D were the most effective interferon subtypes in human corneal cells. Different strains of HSV had different susceptibilities to the various IFN subtypes tested.

Recombinant human interferon alpha D in HSV-1 recurrence in the rabbit.

Recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing the shedding of herpes simplex virus type-1 (HSV-1) induced by 6-hydroxydopamine iontophoresis followed by topical epinephrine application in previously infected rabbit corneas. A treatment schedule of RIFN alpha D, two drops QID was superior to one drop BID. RIFN alpha A also appeared to be effective in reducing viral shedding. Rabbits treated with RIFN alpha D during two episodes of adrenergically induced HSV-1 shedding, but not during anticipated episodes of spontaneous shedding, did not show a significant reduction in shedding of virus. Interferon was present in significantly higher concentration in tear samples following treatment with RIFN alpha D as compared with RIFN alpha A.

Use of recombinant interferon to prevent recurrent herpesvirus shedding.

Iontophoresis with 6-hydroxydopamine was performed in rabbits previously infected with herpes simplex virus, McKrae strain. Viral shedding into the tear film was significantly decreased by the use of recombinant alpha interferon subtype D given as one drop qid. Interferon was noted in the tear film of rabbits 16-18 hours after the last placement of interferon drops into the inferior cul-de-sac.