The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity

<p>Abstract</p> <p>Background</p> <p>HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ.</p> <p>Methods</p> <p>The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. <it>In vivo </it>studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions.</p> <p>Results</p> <p>The gefitinib and RAD001 combination inhibited cell growth <it>in vitro </it>in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated <it>in vitro </it>with cell line dependent increases in cytotoxicity and cytostasis while treatment <it>in vivo </it>promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (<it>in vitro </it>and <it>in vivo</it>) and EGFR signaling <it>in vivo </it>relative to the single drugs.</p> <p>Conclusions</p> <p>The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.</p

Role of drug release and liposome-mediated drug delivery in governing the therapeutic activity of liposomal mitoxantrone used to treat human A431 and LS180 solid tumors

ABSTRACT A previous study suggested that drug release is the dominating factor controlling biological activity of liposomal mitoxantrone in tissues where the rate of liposome accumulation is rapid. The studies described here attempted to address the question: under conditions where the rate of liposome accumulation is slow, does drug release or liposome-mediated drug delivery become the dominant factor controlling therapeutic activity? Liposomal mitoxantrone formulations exhibiting different drugrelease characteristics were injected i.v. in mice bearing human carcinoma xenografts: A431 human squamous cell carcinoma and LS180 human colon cell carcinoma in SCID/RAG 2 mice. When lipid and drug levels were measured in established (Ͼ100-mg) tumors, accumulation was more rapid in the LS180 tumors (C max 4 h) than in the A431 tumors (C max 48 h). Mean area under the curve values for mitoxantrone measured over a 96-h time course in A431 tumors were 505, 304, and 93 g ⅐ g Ϫ1 ⅐ h Ϫ1 for 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/Chol, and free mitoxantrone, respectively. When a similar analysis was completed in LS180 tumors, the area under the curve values were 999, 749, and 251 g ⅐ g Ϫ1 ⅐ h Ϫ

Combined RNAi-mediated suppression of Rictor and EGFR resulted in complete tumor regression in an orthotopic glioblastoma tumor model.

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM

Vascular normalization in orthotopic glioblastoma following intravenous treatment with lipid-based nanoparticulate formulations of irinotecan (Irinophore C™), doxorubicin (Caelyx®) or vincristine

Background: Chemotherapy for glioblastoma (GBM) patients is compromised in part by poor perfusion in the tumor. The present study evaluates how treatment with liposomal formulation of irinotecan (Irinophore C™), and other liposomal anticancer drugs, influence the tumor vasculature of GBM models grown either orthotopically or subcutaneously. Methods Liposomal vincristine (2 mg/kg), doxorubicin (Caelyx®; 15 mg/kg) and irinotecan (Irinophore C™; 25 mg/kg) were injected intravenously (i.v.; once weekly for 3 weeks) in Rag2M mice bearing U251MG tumors. Tumor blood vessel function was assessed using the marker Hoechst 33342 and by magnetic resonance imaging-measured changes in vascular permeability/flow (Ktrans). Changes in CD31 staining density, basement membrane integrity, pericyte coverage, blood vessel diameter were also assessed. Results The three liposomal drugs inhibited tumor growth significantly compared to untreated control (p < 0.05-0.001). The effects on the tumor vasculature were determined 7 days following the last drug dose. There was a 2-3 fold increase in the delivery of Hoechst 33342 observed in subcutaneous tumors (p < 0.001). In contrast there was a 5-10 fold lower level of Hoechst 33342 delivery in the orthotopic model (p < 0.01), with the greatest effect observed following treatment with Irinophore C. Following treatment with Irinophore C, there was a significant reduction in Ktrans in the orthotopic tumors (p < 0.05). Conclusion The results are consistent with a partial restoration of the blood-brain barrier following treatment. Further, treatment with the selected liposomal drugs gave rise to blood vessels that were morphologically more mature and a vascular network that was more evenly distributed. Taken together the results suggest that treatment can lead to normalization of GBM blood vessel the structure and function. An in vitro assay designed to assess the effects of extended drug exposure on endothelial cells showed that selective cytotoxic activity against proliferating endothelial cells could explain the effects of liposomal formulations on the angiogenic tumor vasculature.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofPharmaceutical Sciences, Faculty ofOther UBCNon UBCReviewedFacult

Induction of lentiviral shRNA-transduced cells results in downregulation of corresponding proteins <i>in vitro</i> and downstream effectors, and reduction in cell migration.

<p><b>a</b>) Representative immunoblots showing Rictor, EGFR and β-actin from parental U251MG cells, U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor in the absence (-) or presence (+) of doxycycline. Average of band optical density normalized to β-actin from three independent experiments (+/−SEM), and expressed as relative to values obtained from parental cells, is shown under each band. *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to parental cells. <b>b</b>) Representative immunoblots showing Rictor, EGFR, p(473)-AKT, p(346)-NDRG1, p(422)-SGK, p(657)-PKCα and β-actin from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor exposed to doxycycline for 72hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x is shown under each band. <b>c</b>) Representative immunoblots showing Rictor, p(473)-AKT and β-actin from U251^Rictor in the absence (-) of doxycycline or exposed to doxycycline for 24–120 hrs. Average of band optical density normalized to β-actin and expressed as relative to values obtained from U251^Ng2x in the absence of doxycycline is shown under each band. <b>d</b>) Scratch width scoring of U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor 18hrs after scratching in presence of doxycycline and after pre-incubation with doxycycline for 72 hrs.**p-value ≤0.01 compared to U251^Ng2x cells.</p

Transfection of siRNA sequences specific to Rictor and EGFR results in downregulation of their respective proteins in U251MG cell line.

<p>Optical density values shown are expressed relative to values obtained from untreated cells, which correspond to a value of 1. <b>a</b>) Representative immunoblots showing ILK, Rictor, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against ILK or Rictor or the negative control sequence (Ng ctrl). <b>b</b>) Representative immunoblots showing EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96hrs after transfection of siRNA against EGFR or the negative control sequence (Neg ctrl). <b>c</b>) Representative immunoblots showing Rictor, EGFR, p(473)AKT, AKT and β-actin from U251MG cells 96 hrs after transfection of the combination of Rictor and EGFR siRNAs or the combination of two negative control sequences (Ng2x). Optical density values shown under each band represent the average obtained from three independent experiments (±SEM) normalized to β-actin, and AKT in the case of p(473)AKT. <b>d</b>) Representative fluorescence photomicrograph (n = 3) of U251MG cells showing nuclei (Draq5; red), F-actin (Texas red phalloidin; Yellow), and p(473)-AKT (Alexa 488; blue) 96 hrs after transfection of siRNA against Rictor, EGFR, the combination of Rictor and EGFR, or the combination of two negative sequences (Ng2x).</p

Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.

<p>Fluorescence micrographs showing EGFR (Alexa 488; green), Rictor (Alexa 488; yellow) and cell nuclei (Hoechst 33342; blue) in GBM4 GBM-derived cancer stem-like cell line, and Gli36, U251MG, U118MG and LN229 GBM cell lines.</p

The combined silencing of Rictor and EGFR <i>in vivo</i> results in a complete inhibition of tumor growth.

<p>U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expression in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. <b>a</b>) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251^EGFR/Rictor group. <b>b</b>) A representative brain section from U251^Ng2x, U251^Rictor, U251^EGFR and U251^EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). <b>c</b>) A representative tumor section from U251^Ng2x, U251^Rictor and U251^EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). <b>d</b>) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251^Ng2x, U251^Rictor, U251^EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). <b>e</b>) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251^EGFR/Rictor group.</p