Additional file 1 of Combination of GT90001 and nivolumab in patients with advanced hepatocellular carcinoma: a multicenter, single-arm, phase 1b/2 study

Additional file 1: Appendix Table 1. Complete eligibility criteria. Appendix Table S2. Details of dose reduction or interruption due to treatment-emergent adverse events in all patients. Appendix Figure S1. Study procedures. Appendix Figure S2. Changes in patient blood pressure. (A) Changes in blood pressure between baseline and at the end of study drug treatment. (B) The percentage of patients who experienced a change in blood pressure. Abbreviations: SBP=systolic blood pressure; DBP=diastolic blood pressure

Presentation_1_Case Report: Maintenance Nivolumab in Complete Responder After Multimodality Therapy in Metastatic Pancreatic Adenocarcinoma.pptx

Maintenance therapy is rarely considered in pancreatic ductal adenocarcinoma (PDAC). We describe the case of a 57-year-old man with metastatic PDAC treated with an initially full but subsequently de-escalated dose of combination chemotherapy due to intolerance to neurotoxicity. After a complete response to combined radiofrequency ablation for the liver metastasis and radiotherapy for the pancreatic tumor was achieved, chemotherapy was terminated and maintenance therapy was applied: nivolumab plus cytokine-induced killer cell therapy initially and then a de-escalated dosing interval of nivolumab monotherapy subsequently. No adverse events occurred during nivolumab therapy for more than 2 years, and the patient remains disease-free. To date, this is the first report of maintenance nivolumab after successful multimodality therapy in metastatic PDAC.</p

Supplementary materials and methods, supplement figure S1: to S12, and supplement table S1 from Cyclin E1 Inhibition can Overcome Sorafenib Resistance in Hepatocellular Carcinoma Cells Through Mcl-1 Suppression

Supplement Figure S1: The six E2Fs binding sites in cyclin E1 promoter; Supplement Figure S2: Suppression of cyclin E1 by sorafenib correlated with sorafenib sensitivity; Supplement Figure S3: Effects of sorafenib and the Raf kinase inhibitor on cyclin E1 suppression in HCC cells; Supplement Figure S4: Knockdown of cyclin E1, but not cyclin E2, significantly increased the sensitivity to sorafenib in HCC cells; Supplement Figure S5: Effects of cyclin E1 or Mcl-1 inhibition on sorafenib-induced apoptosis; Supplement Figure S6: Effects of E2F1 suppression on cyclin E1 expression; Supplement Figure S7: Effects of flavopiridol on cyclin E1 and Rb protein levels in HCC cells; Supplement Figure S8: Synergistic anti-tumor activity between sorafenib and the CDK inhibitor flavopiridol; Supplement Figure S9: Differential effect between sorafenib and the CDK inhibitor flavopiridol on cell-cycle progression of HCC cells; Supplement Figure S10: The apoptosis-related proteins measured by the Human Apoptosis Array kit; Supplement Figure S11: In vivo evidence of toxicity between sorafenib and flavopiridol in HCC cells; Supplement Figure S12: Flavopiridol enhanced the anti-tumor efficacy of sorafenib in vivo; and Supplement Table S1: Primers list and Supplementary materials and methods.</p

Effects of IGFR signaling modulation on drug resistance of HCC cells.

<p>(A) Addition of IGF1 or IGF2 (100 ng/ml) into the culture medium can activate IGFR and AKT signaling in all the HCC cells (Hep3B, SK-Hep1, and PLC5) and HUVEC. The activation can be inhibited by the IGFR inhibitor NVP-AEW54. (B) Growth curves of HCC cells and HUVEC with or without stimulation by IGF1 or IGF2. Cell viability was assessed by MTT assay. Points, mean (n = 3); bars, SD<b>.</b> (C) Percentage of apoptotic cells was measured by flow cytometry after 72 hours of drug treatment in Hep3B and HUVEC. **, p<0.05 compared with cells untreated with IGF2. (D) Effects of IGF stimulation on sorafenib, and sunitinib on P ARP-1 cleavage and caspase 3 activation.</p

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

<div><p>Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.</p></div

In vivo anti-tumor synergy between IGFR inhibition and other MTAs.

<p>Hep3B cells were injected subcutaneously into male BALB/c athymic nude mice. Mice were treated daily by gavage as indicated (NVP-30, NVP-AEW541 30 mg/kg/day; Sor-10, sorafenib 10 mg/kg/day; Sun-40, sunitinib 40 mg/kg/day, animal number n = 5 in each treatment group). Difference in (A) tumor growth; (B) tumor cell apoptosis (TUNEL assay).**, P<0.01, compared with control (vehicle treatment). (C) Difference in Chk2 phosphorylation measured by Western blotting.</p

FOXO3a decreases expression of ER-regulated genes and increases CDK inhibitors in MCF7-FO breast tumors

At 35 days after tumor cell implantation, breast tumors derived from female athymic mice bearing MCF7-FO or MCF7-C (control) tumors were resected, fixed, sectioned, and placed on slides. Five independent tumors (each from a different mouse) were tested in each mouse group. Tumor specimens were subjected to immunohistochemical (IHC) staining with antibodies specific to forkhead box class O (FOXO)3a, hemagglutinin (HA)-tag, pS2, complement C3, cathepsin (Cath)-D, progesterone receptor (PgR), and cyclin D. Slides were examined at 40× magnification with a microscope. Numbers of positively staining cells in four random fields were counted in each tumor section, and representative fields are shown. Scale bars indicate 50 μm. *< 0.01 between control MCF7-C group versus MCF7-FO group. At 35 days after tumor cell implantation, breast tumors derived from female athymic mice bearing MCF7-FO or MCF7-C (control) tumors were resected, fixed, sectioned, and placed on slides. Five independent tumors (each from a different mouse) were tested in each mouse group. Tumor specimens were subjected to IHC staining with antibodies to p21Cip1, p27Kip1, p57Kip2, estrogen receptor (ER)-α, and ER-β. Numbers of positively staining cells in four random fields were counted in each tumor section, and representative fields are shown. Scale bars indicate 50 μm. *< 0.02 between control MCF7-C group versus MCF7-FO group. Confirmation that FOXO3a enhances expression of cyclin-dependent kinase (CDK) inhibitors and reduces expression of cyclin Din tumors in immunoblotting (IB) analysis. Whole lysates of MCF7-FO or MCF7-C breast tumor specimens were subjected to IB analysis with antibodies to p21Cip1, p27Kip1, p57Kip2, cyclin D, ER-α, ER-β, HA-tag and FOXO3a (positive controls), and β-actin (loading control).<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p

p-Chk2 is a possible mediator of anti-tumor synergy between IGFR inhibition and sunitinib.

<p>(A) Effects between NVP-AEW541, sorafenib, and sunitinib on apoptosis-related proteins in HCC cells. Hep3B cells were treated with NVP-AEW541, sorafenib, and sunitinib at the indicated concentrations for 48 h. Whole-cell lysates were subjected to Western blotting. (B and C) Hep3B cells were transfected with si-Chk2 or scrambled siRNA for 24 hours, and then treated with the indicated drugs. Whole-cell lysates were collected for Western blotting after 48 h drug treatment. The percentages of apoptotic cells were measured by flow cytometry after 72 h drug treatment. **, p<0.01.</p

Synergistic growth-inhibitory and apoptosis-inducing effects between IGFR inhibition and other MTAs.

<p>(A) Median dose effect analysis of synergistic growth inhibitory effects. Growth inhibition was measured by MTT assay. CI was calculated using the CI-isobologram method. CI = 1, additive effect; CI <1, synergistic effect; CI >1, antagonistic effect. (B and C) Synergistic apoptosis-inducing effects between IGFR inhibition and other MTAs in Hep3B cells measured by flow cytometry (sub-G1 fraction analysis) (B) and by PARP-1 cleavage and caspase 3 activation (C). **, p<0.05 compared with cells not treated with the IGFR inhibitor NVP-AEW541. (D) IGFR inhibition by siRNA knockdown enhanced the apoptosis-inducing effects of sunitinib, but not sorafenib or NVP-AEW541, in Hep3B cells.</p

FOXO3a regulates expression of ER target genes and CDK inhibitors, and induces apoptosis in MCF-7

Ectopic expression of Forkhead box class O (FOXO)3a reduces the expression of some estrogen receptor (ER)-regulated genes and enhances the expression of cyclin-dependent kinase (CDK) inhibitors in MCF7-FO cells in the presence of 17β-estradiol (E2). Immunoblotting (IB) analyses for HA-FOXO3a, endogenous FOXO3a, p27Kip1, p21Cip1, p57Kip2, cyclin D, cyclin E, cathepsin D, progesterone receptor (PgR), ER-α, and ER-β protein expression in MCF7-FO33 and MCF7-FO41 cells (constitutively expressing FOXO3a) and in control (MCF-7 and MCF7-C5) cells were performed with specific antibodies, as indicated. Equal loading was confirmed by the same IB analysis with antibodies against β-actin or β-tubulin. MDA-MB-453 (MDA-453, ER-negative) cells were transfected with either FOXO3a or an empty pCDNA3.1 vector (control), as indicated. Total lysates of the transfected cells were prepared and subjected to SDS-PAGE followed by IB analysis with the indicated antibodies.<p><b>Copyright information:</b></p><p>Taken from "Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis"</p><p>http://breast-cancer-research.com/content/10/1/R21</p><p>Breast Cancer Research : BCR 2008;10(1):R21-R21.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2374977.</p><p></p