The development of drug resistance by cancer cells is recognized as a major cause for drug failure and disease progression. The PI3K/AKT/mTOR pathway is aberrantly stimulated in many cancer cells and thus it has emerged as a target for therapy. However, mTORC1 and S6K also mediate potent negative feedback loops that attenuate signaling via insulin/insulin growth factor receptor and other tyrosine kinase receptors. Suppression of these feedback loops causes overactivation of upstream pathways, including PI3K, AKT, and ERK that potentially oppose the antiproliferative effects of mTOR inhibitors and lead to drug resistance. A corollary of this concept is that release of negative feedback loops and consequent compensatory overactivation of promitogenic pathways in response to signal inhibitors can circumvent the mitogenic block imposed by targeting only one pathway. Consequently, the elucidation of the negative feedback loops that regulate the outputs of signaling networks has emerged as an area of fundamental importance for the rational design of effective anticancer combinations of inhibitors. Here, we review pathways that undergo compensatory overactivation in response to inhibitors that suppress feedback inhibition of upstream signaling and underscore the importance of unintended pathway activation in the development of drug resistance to clinically relevant inhibitors of mTOR, AKT, PI3K, or PI3K/mTOR. Mol Cancer Ther; 13(11); 2477–88. ©2014 AACR.
Despite promising preclinical results with mTOR kinase inhibitors in multiple myeloma, resistance to these drugs may arise via feedback activation loops. This concern is especially true for insulin-like growth factor 1 receptor (IGF1R), because IGF1R signaling is downregulated by multiple AKT and mTOR feedback mechanisms. We have tested this hypothesis in multiple myeloma using the novel selective mTOR kinase inhibitor AZD8055. We evaluated p-mTOR S2481 as the readout for mTORC2/Akt activity in multiple myeloma cells in the context of mTOR inhibition via AZD8055 or rapamycin. We next validated AZD8055 inhibition of mTORC1 and mTORC2 functions in multiple myeloma cells alone or in culture with bone marrow stroma cells and growth factors. Unlike rapamycin, AZD8055 resulted in apoptosis of multiple myeloma cells. AZD8055 treatment, however, induced upregulation of IGF1R phosphorylation in p-Akt S473–expressing multiple myeloma cell lines. Furthermore, exposure of AZD8055-treated cells to IGF1 induced p-Akt S473 and rescued multiple myeloma cells from apoptosis despite mTOR kinase inhibition and TORC2/Akt blockage. The addition of blocking IGF1R antibody resulted in reversing this effect and increased AZD8055-induced apoptosis. Our study suggests that combination treatment with AZD8055 and IGF1R-blocking agents is a promising strategy in multiple myeloma with potential IGF1R/Akt signaling–mediated survival. Mol Cancer Ther; 13(11); 2489–500. ©2014 AACR.
Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcome and few validated drug targets. Two prevalent features of TNBC, tumor hypoxia and derangement of homologous recombination (HR) repair, are potentially exploitable for therapy. This study investigated whether hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may inhibit growth of TNBC by simultaneously addressing these two targets. We measured in vitro activity of HAP of DNA breakers (tirapazamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in TNBC cell lines and isogenic models, and related this to measures of HR repair and expression of prodrug-activating enzymes. Antitumor activity of HAP was examined in isogenic BRCA2-knockout xenograft models and compared with platinum chemotherapy. All five HAP selectively inhibited growth of TNBC cell lines under hypoxia. Sensitivity to HAP was not strongly associated with BRCA1 genotype. However, HAP sensitivity was enhanced by suppression of HR (assessed by radiation-induced RAD51 focus formation) when BRCA1 and PALB2 were knocked down in a common (MDA-MB-231) background. Furthermore, knockout of BRCA2 markedly sensitized DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and PR-104 and significantly augmented sterilization of clonogens by these agents in xenografts, both as monotherapy and in combination with radiotherapy, but had less effect on activity of the benzotriazine di-N-oxide SN30000. PR-104 monotherapy was more effective than cisplatin at inhibiting growth of BRCA2-knockout tumors at equitoxic doses. This study demonstrates the potential for HAP of nitrogen mustards to simultaneously exploit hypoxia and HR defects in tumors, with translational implications for TNBC and other HR-deficient malignancies. Mol Cancer Ther; 13(11); 2501–14. ©2014 AACR.
Endocrine-resistant breast cancer is a major clinical obstacle. The use of 17β-estradiol (E2) has reemerged as a potential treatment option following exhaustive use of tamoxifen or aromatase inhibitors, although side effects have hindered its clinical usage. Protein kinase C alpha (PKCα) expression was shown to be a predictor of disease outcome for patients receiving endocrine therapy and may predict a positive response to an estrogenic treatment. Here, we have investigated the use of novel benzothiophene selective estrogen mimics (SEM) as an alternative to E2 for the treatment of tamoxifen-resistant breast cancer. Following in vitro characterization of SEMs, a panel of clinically relevant PKCα-expressing, tamoxifen-resistant models were used to investigate the antitumor effects of these compounds. SEM treatment resulted in growth inhibition and apoptosis of tamoxifen-resistant cell lines in vitro. In vivo SEM treatment induced tumor regression of tamoxifen-resistant T47D:A18/PKCα and T47D:A18-TAM1 tumor models. T47D:A18/PKCα tumor regression was accompanied by translocation of estrogen receptor (ER) α to extranuclear sites, possibly defining a mechanism through which these SEMs initiate tumor regression. SEM treatment did not stimulate growth of E2-dependent T47D:A18/neo tumors. In addition, unlike E2 or tamoxifen, treatment with SEMs did not stimulate uterine weight gain. These findings suggest the further development of SEMs as a feasible therapeutic strategy for the treatment of endocrine-resistant breast cancer without the side effects associated with E2. Mol Cancer Ther; 13(11); 2515–26. ©2014 AACR.
Pazopanib is an orally bioavailable, ATP-competitive, multitargeted tyrosine kinase inhibitor mainly targeting VEGFR2 and PDGFR tyrosine kinases, but the biologic sequences of pazopanib activities beyond antiangiogenesis are poorly defined. We used a panel of 38 gastric cancer cell lines to test the efficacy of pazopanib. In a growth inhibition assay, genomic changes indicated that pazopanib had differential effects on cell growth. Treatment of the KATO-III, OCUM-2M, SNU-16, and HSC-39 gastric cancer cell lines harboring FGFR2 amplification with pazopanib resulted in marked decreases of cell survival with IC50 in ranges of 0.1 to 2.0 μmol/L, whereas the same treatment of those cell lines without FGFR2 amplification had no growth-inhibitory effects. In the ectopic FGFR2-expressing model, treatment with the indicated concentrations of pazopanib significantly inhibited cell growth and colony formation by FGFR2-expressing NIH 3T3 cells with wild-type (WT) FGFR2 and mutant FGFR2 (S252W). Pazopanib also selectively suppressed constitutive FGFR2 signaling and phosphorylation of downstream effectors. In cell-cycle analysis, FGFR2-amplified cells underwent cell-cycle arrest at the G1–S phase after pazopanib treatment, whereas there were no significant effects on cell-cycle progression in cells without FGFR2 amplification treated with pazopanib. In addition, pazopanib increased a substantial fraction of sub-G1 only in FGFR2-amplified cells. These findings show that the activation of FGFR2 signaling by amplification may be a critical mediator of cell proliferation in a small subset of gastric cancer patients and that pazopanib may provide genotype-correlated clinical benefits beyond the setting of highly vascular tumors. Mol Cancer Ther; 13(11); 2527–36. ©2014 AACR.
Activation of the p53 pathway has been considered a therapeutic strategy to target cancers. We have previously identified several p53-activating small molecules in a cell-based screen. Two of the compounds activated p53 by causing DNA damage, but this modality was absent in the other four. We recently showed that one of these, BMH-21, inhibits RNA polymerase I (Pol I) transcription, causes the degradation of Pol I catalytic subunit RPA194, and has potent anticancer activity. We show here that three remaining compounds in this screen, BMH-9, BMH-22, and BMH-23, cause reorganization of nucleolar marker proteins consistent with segregation of the nucleolus, a hallmark of Pol I transcription stress. Further, the compounds destabilize RPA194 in a proteasome-dependent manner and inhibit nascent rRNA synthesis and expression of the 45S rRNA precursor. BMH-9– and BMH-22–mediated nucleolar stress was detected in ex vivo–cultured human prostate tissues indicating good tissue bioactivity. Testing of closely related analogues showed that their activities were chemically constrained. Viability screen for BMH-9, BMH-22, and BMH-23 in the NCI60 cancer cell lines showed potent anticancer activity across many tumor types. Finally, we show that the Pol I transcription stress by BMH-9, BMH-22, and BMH-23 is independent of p53 function. These results highlight the dominant impact of Pol I transcription stress on p53 pathway activation and bring forward chemically novel lead molecules for Pol I inhibition, and, potentially, cancer targeting. Mol Cancer Ther; 13(11); 2537–46. ©2014 AACR.
The FGF receptors (FGFR) are tyrosine kinases that are constitutively activated in a subset of tumors by genetic alterations such as gene amplifications, point mutations, or chromosomal translocations/rearrangements. Recently, small-molecule inhibitors that can inhibit the FGFR family as well as the VEGF receptor (VEGFR) or platelet-derived growth factor receptor (PDGFR) family displayed clinical benefits in cohorts of patients with FGFR genetic alterations. However, to achieve more potent and prolonged activity in such populations, a selective FGFR inhibitor is still needed. Here, we report the identification of CH5183284/Debio 1347, a selective and orally available FGFR1, FGFR2, and FGFR3 inhibitor that has a unique chemical scaffold. By interacting with unique residues in the ATP-binding site of FGFR1, FGFR2, or FGFR3, CH5183284/Debio 1347 selectively inhibits FGFR1, FGFR2, and FGFR3 but does not inhibit kinase insert domain receptor (KDR) or other kinases. Consistent with its high selectivity for FGFR enzymes, CH5183284/Debio 1347 displayed preferential antitumor activity against cancer cells with various FGFR genetic alterations in a panel of 327 cancer cell lines and in xenograft models. Because of its unique binding mode, CH5183284/Debio 1347 can inhibit FGFR2 harboring one type of the gatekeeper mutation that causes resistance to other FGFR inhibitors and block FGFR2 V564F–driven tumor growth. CH5183284/Debio 1347 is under clinical investigation for the treatment of patients harboring FGFR genetic alterations. Mol Cancer Ther; 13(11); 2547–58. ©2014 AACR.
Targeting the EGFR, with inhibitors such as erlotinib, represents a promising therapeutic option in advanced head and neck squamous cell carcinomas (HNSCC). However, they lack significant efficacy as single agents. Recently, we identified the ability of statins to induce synergistic cytotoxicity in HNSCC cells through targeting the activation and trafficking of the EGFR. However, in a phase I trial of rosuvastatin and erlotinib, statin-induced muscle pathology limited the usefulness of this approach. To overcome these toxicity limitations, we sought to uncover other potential combinations using a 1,200 compound screen of FDA-approved drugs. We identified monensin, a coccidial antibiotic, as synergistically enhancing the cytotoxicity of erlotinib in two cell line models of HNSCC, SCC9 and SCC25. Monensin treatment mimicked the inhibitory effects of statins on EGFR activation and downstream signaling. RNA-seq analysis of monensin-treated SCC25 cells demonstrated a wide array of cholesterol and lipid synthesis genes upregulated by this treatment similar to statin treatment. However, this pattern was not recapitulated in SCC9 cells as monensin specifically induced the expression of activation of transcription factor (ATF) 3, a key regulator of statin-induced apoptosis. This differential response was also demonstrated in monensin-treated ex vivo surgical tissues in which HMG-CoA reductase expression and ATF3 were either not induced, induced singly, or both induced together in a cohort of 10 patient samples, including four HNSCC. These results suggest the potential clinical utility of combining monensin with erlotinib in patients with HNSCC. Mol Cancer Ther; 13(11); 2559–71. ©2014 AACR.
We previously identified a novel MLL5 isoform, MLL5β, which was essential for E6 and E7 transcriptional activation in HPV16/18-associated cervical cancers. In this report, we investigated the potential of RNAi-mediated silencing of MLL5β through the use of MLL5β-siRNA as a novel therapeutic strategy for HPV16/18-positive cervical cancer. We observed concurrent downregulation of E6 and E7 after MLL5β silencing, leading to growth inhibition via the activation of apoptosis and senescence in the HeLa cell model. This corresponded with the enhanced antitumor effects of MLL5β-siRNA compared with E6- or E7-siRNA single treatments. Significant reduction in tumor size after MLLβ-siRNA treatment in the HeLa xenograft tumor model further emphasized the importance of MLL5β in HPV16/18-associated tumor growth and the potential of RNAi therapeutics that target MLL5β. We also identified MLL5β as a modulator of gamma-irradiation (IR) sensitization properties of cisplatin. We observed that while MLL5β silencing alone was enough to evoke cisplatin-like IR sensitization in tumor cells in vitro, overexpression of MLL5β inhibited the ability of cisplatin to sensitize HeLa cells to IR-induced cytotoxicity. MLL5β-siRNA-IR cotreatment was also observed to enhance tumor growth inhibition in vivo. Taken together, our findings highlight the potential of targeted silencing of MLL5β via the use of MLL5β-siRNA as a novel therapeutic strategy and propose that MLL5β-siRNA could be a viable alternative for cisplatin in the current cisplatin-based chemotherapeutics for HPV16/18-associated cervical cancers. Mol Cancer Ther; 13(11); 2572–82. ©2014 AACR.
Pancreatic stellate cells (PSC) have been recognized as the principal cells responsible for the production of fibrosis in pancreatic ductal adenocarcinoma (PDAC). Recently, PSCs have been noted to share characteristics with cells of monocyte-macrophage lineage (MML cells). Thus, we tested whether PSCs could be targeted with the nitrogen-containing bisphosphonates (NBP; pamidronate or zoledronic acid), which are potent MML cell inhibitors. In addition, we tested NBPs treatment combination with nanoparticle albumin–bound paclitaxel (nab-paclitaxel) to enhance antitumor activity. In vitro, we observed that PSCs possess α-naphthyl butyrate esterase (ANBE) enzyme activity, a specific marker of MML cells. Moreover, NBPs inhibited PSCs proliferation, activation, release of macrophage chemoattractant protein-1 (MCP-1), and type I collagen expression. NBPs also induced PSCs apoptosis and cell-cycle arrest in the G1 phase. In vivo, NBPs inactivated PSCs; reduced fibrosis; inhibited tumor volume, tumor weight, peritoneal dissemination, angiogenesis, and cell proliferation; and increased apoptosis in an orthotopic murine model of PDAC. These in vivo antitumor effects were enhanced when NBPs were combined with nab-paclitaxel but not gemcitabine. Our study suggests that targeting PSCs and tumor cells with NBPs in combination with nab-paclitaxel may be a novel therapeutic approach to PDAC. Mol Cancer Ther; 13(11); 2583–94. ©2014 AACR.
Prostate cancer is the most common noncutaneous malignancy affecting men in North America. Radical prostatectomy remains a definitive treatment for prostate cancer. However, prostate surgeries are still performed "blindly" with the extent of tumor infiltration past the margins of the surgery only being determined postoperatively. An imaging modality that can be used during surgery is needed to help define the tumor margins. With its abundant expression in prostate cancer, prostate-specific membrane antigen (PSMA) is an ideal target for detection of prostate cancer. The purpose of this study was to develop PSMA-targeted near-infrared (NIR) optical imaging probes for intraoperative visualization of prostate cancer. We synthesized a high-affinity PSMA ligand (PSMA-1) with low molecular weight and further labeled it with commercially available NIR dyes IRDy800 and Cy5.5. PSMA-1 and PSMA-1–NIR conjugates had binding affinities better than the parent ligand Cys-CO-Glu. Selective binding was measured for each of the probes in both in vitro and in vivo studies using competitive binding and uptake studies. Interestingly, the results indicated that the pharmacokinetics of the probes was dependent of the fluorophore conjugated to the PSMA-1 ligand and varied widely. These data suggest that PSMA-targeted probes have the potential to be further developed as contrast agents for clinical intraoperative fluorescence-guided surgery. Mol Cancer Ther; 13(11); 2595–606. ©2014 AACR.
MET, the receptor of hepatocyte growth factor, plays important roles in tumorigenesis and drug resistance in numerous cancers, including non–small cell lung cancer (NSCLC). As increasing numbers of MET inhibitors are being developed for clinical applications, antibody fragment–based immunopositron emission tomography (immunoPET) has the potential to rapidly quantify in vivo MET expression levels for drug response evaluation and patient stratification for these targeted therapies. Here, fully human single-chain variable fragments (scFvs) isolated from a phage display library were reformatted into bivalent cys-diabodies (scFv-cys dimers) with affinities to MET ranging from 0.7 to 5.1 nmol/L. The candidate with the highest affinity, H2, was radiolabeled with 89Zr for immunoPET studies targeting NSCLC xenografts: low MET-expressing Hcc827 and the gefitinib-resistant Hcc827-GR6 with 4-fold MET overexpression. ImmunoPET at as early as 4 hours after injection produced high-contrast images, and ex vivo biodistribution analysis at 20 hours after injection showed about 2-fold difference in tracer uptake levels between the parental and resistant tumors (P < 0.01). Further immunoPET studies using a larger fragment, the H2 minibody (scFv-CH3 dimer), produced similar results at later time points. Two of the antibody clones (H2 and H5) showed in vitro growth inhibitory effects on MET-dependent gefitinib-resistant cell lines, whereas no effects were observed on resistant lines lacking MET activation. In conclusion, these fully human antibody fragments inhibit MET-dependent cancer cells and enable rapid immunoPET imaging to assess MET expression levels, showing potential for both therapeutic and diagnostic applications. Mol Cancer Ther; 13(11); 2607–17. ©2014 AACR.
A linker-drug platform was built on the basis of a cleavable linker-duocarmycin payload for the development of new-generation antibody–drug conjugates (ADC). A leading ADC originating from that platform is SYD983, a HER2-targeting ADC based on trastuzumab. HER2-binding, antibody-dependent cell-mediated cytotoxicity and HER2-mediated internalization are similar for SYD983 as compared with trastuzumab. HER2-expressing cells in vitro are very potently killed by SYD983, but SYD983 is inactive in cells that do not express HER2. SYD983 dose dependently reduces tumor growth in a BT-474 mouse xenograft in vivo. The ADC is stable in human and cynomolgus monkey plasma in vitro but shows relatively poor stability in mouse plasma due to mouse-specific carboxylesterase. SYD983 could be dosed up to 30 mg/kg in cynomolgus monkeys with high exposure, excellent stability in blood, and without severe toxic effects. The monkey safety study showed no SYD983-induced thrombocytopenia and no induction of peripheral sensory neuropathy, both commonly observed in trials and studies with ADCs based on tubulin inhibitors. Finally, to improve homogeneity, SYD983 was further purified by hydrophobic interaction chromatography resulting in an ADC (designated SYD985) predominantly containing DAR2 and DAR4 species. SYD985 showed high antitumor activity in two patient-derived xenograft models of HER2-positive metastatic breast cancers. In conclusion, the data obtained indicate great potential for this new HER2-targeting ADC to become an effective drug for patients with HER2-positive cancers with a favorable safety profile. More generally, this new-generation duocarmycin-based linker-drug technology could be used with other mAbs to serve more indications in oncology. Mol Cancer Ther; 13(11); 2618–29. ©2014 AACR.
Mesothelin (MSLN) is an attractive target for antibody–drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine–citrulline linker. The humanized (h7D9.v3) version, αMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of αMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose-dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of αMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial. Mol Cancer Ther; 13(11); 2630–40. ©2014 AACR.
The combination of immunostimulatory agents with cytotoxic drugs is emerging as a promising approach for potentially curative tumor therapy, but advances in this field are hindered by the requirement of testing individual combination partners as single agents in dedicated clinical studies, often with suboptimal efficacy. Here, we describe for the first time a novel multipayload class of targeted drugs, the immunocytokine–drug conjugates (IDC), which combine a tumor-homing antibody, a cytotoxic drug, and a proinflammatory cytokine in the same molecular entity. In particular, the IL2 cytokine and the disulfide-linked maytansinoid DM1 microtubular inhibitor could be coupled to the F8 antibody, directed against the alternatively spliced EDA domain of fibronectin, in a site-specific manner, yielding a chemically defined product with selective tumor-homing performance and potent anticancer activity in vivo, as tested in two different immunocompetent mouse models. Mol Cancer Ther; 13(11); 2641–52. ©2014 AACR.
The RG7787 mesothelin-targeted recombinant immunotoxin (RIT) consists of an antibody fragment targeting mesothelin (MSLN) fused to a 24-kD fragment of Pseudomonas exotoxin A for cell killing. Compared with prior RITs, RG7787 has improved properties for clinical development including decreased nonspecific toxicity and immunogenicity and resistance to degradation by lysosomal proteases. MSLN is a cell surface glycoprotein highly expressed by many solid tumor malignancies. New reports have demonstrated that MSLN is expressed by a significant percentage of triple-negative breast and gastric cancer clinical specimens. Here, panels of triple-negative breast and gastric cancer cell lines were tested for surface MSLN expression, and for sensitivity to RG7787 in vitro and in animal models. RG7787 produced >95% cell killing of the HCC70 and SUM149 breast cancer cell lines in vitro with IC50 < 100 pmol/L. RG7787 was also effective against gastric cancer cell lines MKN28, MKN45, and MKN74 in vitro, with subnanomolar IC50s. In a nude mouse model, RG7787 treatment (2.5 mg/kg i.v. qod x3–4) resulted in a statistically significant 41% decrease in volumes of HCC70 xenograft tumors (P < 0.0001) and an 18% decrease in MKN28 tumors (P < 0.0001). Pretreatment with paclitaxel (50 mg/kg i.p.) enhanced efficacy, producing 88% and 70% reduction in tumor volumes for HCC70 and MKN28, respectively, a statistically significant improvement over paclitaxel alone (P < 0.0001 for both). RG7787 merits clinical testing for triple-negative breast and gastric cancers. Mol Cancer Ther; 13(11); 2653–61. ©2014 AACR.
MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma. Mol Cancer Ther; 13(11); 2662–73. ©2014 AACR.
Angiogenesis is required for tumor growth and metastasis, and targeting angiogenesis is a novel anticancer strategy. However, cancer development is a complex multistep process, and single antiangiogenic agents have limited therapeutic efficacy. Here, we report a triple fusion protein, namely CTT peptide–endostatin mimic–kringle 5 (AARP), consisting of MMP-2/9–selective inhibitory peptide (CTT peptide) and well-known endogenous antiangiogenic agents (endostatin mimic and kringle 5), which can simultaneously target matrix metalloproteinases (MMP) and endothelial cells, blocking their actions. AARP was bacterially expressed, and biologic activity of purified AARP was assessed. AARP could significantly inhibit the enzymatic activity of MMP-2/9, proliferation, migration, and tube formation of endothelial cells in vitro. The antitumor activity of AARP was shown in a concentration-dependent manner when injected i.p. into immunodeficient mice bearing multidrug-resistant human epidermoid carcinomas (KB), and AARP is superior to clinical grade endostatin in inhibiting KB xenograft growth. In mouse models of Lewis lung carcinoma (LLC) and hepatoma H22, when given as a single dose, AARP is highly effective for reducing tumor growth, angiogenesis, and metastasis, and increasing survival time. AARP possessed significantly greater antiangiogenic activity than endostatin mimic, CTT peptide–kringle 5 (RK5) both in vitro and in vivo. Compared with conventional chemotherapeutic agents (cyclophosphamide and paclitaxel), AARP is also effective. More importantly, AARP is cytocompatible and no tissue toxicity could be observed after large dose administration. Taken together, our findings suggest AARP is a highly effective, safe, and more potent antiangiogenic agent for blocking tumor angiogenesis and metastasis, and warrants further testing for clinical applications. Mol Cancer Ther; 13(11); 2674–87. ©2014 AACR.
The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)–containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c–related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor–positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications. Mol Cancer Ther; 13(11); 2688–705. ©2014 AACR.
Multidrug resistance (MDR) in cancer is known to decrease the therapeutic efficacy of chemotherapy. The effects of irradiation on MDR in cancer cells remain unclear. Tc-99m methoxyisobutylisonitrile (MIBI) exhibits the same ATP-binding cassette (ABC) transporter kinetics as the chemotherapeutic compound doxorubicin. In this study, we investigated the synergistic effects of chemotherapeutics and irradiation [0 Gy: C (control) group; 3, 6, 9, 12 Gy: I (irradiation) group] in the human non–small lung cancer cell line H1299 exhibiting MDR, on MIBI and doxorubicin ABC transporter kinetics, in vitro and in vivo, respectively. In vitro, inhibition of H1299 cell proliferation by irradiation was found to be irradiation dose dependent. The degree and duration of MDR inhibition in vitro in H1299 were also dose dependent. In the cells of both the C group and 3-Gy I group, no significant difference of MIBI accumulation was observed. In the 6-Gy I group, a higher MIBI accumulation was observed at only 7 days after irradiation relative to the C group. A higher MIBI accumulation in the 9- and 12-Gy I groups with a significant difference from the C group was observed at 4 to 14 days after irradiation. A significant negative correlation between intracellular MIBI accumulation and cell replication was found. In vivo, high accumulation and retention of doxorubicin were observed in irradiated tumors in the H1299 xenograft mice group at 4 to 14 days after 9-Gy irradiation compared with the control mice group. These results provide evidence for a synergistic effect of concurrent chemotherapy and radiotherapy. Mol Cancer Ther; 13(11); 2706–12. ©2014 AACR.
Epithelial-to-mesenchymal transition (EMT) has been associated with poor treatment outcomes in various malignancies and is inversely associated with miRNA145 expression. Therefore, we hypothesized that SNAI2 (Slug) may mediate 5-fluorouracil (5FU) chemotherapy resistance through inhibition of miR145 in colorectal cancer and thus represents a novel therapeutic target to enhance current colorectal cancer treatment strategies. Compared with parental DLD1 colon cancer cells, 5FU-resistant (5FUr) DLD1 cells demonstrated features of EMT, including >2-fold enhanced invasion (P < 0.001) and migration, suppressed E-cadherin expression, and 2-fold increased SNAI2 expression. DLD1 and HCT116 cells with stable expression of SNAI2 (DLD1/SNAI2; HCT116/SNAI2) also demonstrated EMT features such as the decreased E-cadherin as well as significantly decreased miR145 expression, as compared with control empty vector cells. On the basis of an miR145 luciferase promoter assay, we demonstrated that SNAI2 repressed activity of the miR145 promoter in the DLD1 and HCT116 cells. In addition, the ectopic expressing SNAI2 cell lines demonstrated decreased 5FU sensitivity, and, conversely, miR145 replacement significantly enhanced 5FU sensitivity. In the parental SW620 colon cancer cell line with high SNAI2 and low miR145 levels, inhibition of SNAI2 directly with short hairpin sequence for SNAI2 and miR145 replacement therapy both decreased vimentin expression and increased in vitro 5FU sensitivity. In pretreatment rectal cancer patient biopsy samples, low miR145 expression levels correlated with poor response to neoadjuvant 5FU-based chemoradiation. These results suggested that the SNAI2:miR145 pathway may represent a novel clinical therapeutic target in colorectal cancer and may serve as a response predictor to chemoradiation therapy. Mol Cancer Ther; 13(11); 2713–26. ©2014 AACR.
The ability of a panel of camptothecin derivatives to access the tumor compartment was evaluated to determine the mechanisms by which the architecture of solid tumors may act to limit their activity. Microregional localization and activity of members of the camptothecin class of topoisomerase I targeting agents, including topotecan, irinotecan, and irinophore C, a lipid-based nanoparticulate formulation of irinotecan, were evaluated over time in HCT116 and HT29 colorectal tumor xenografts. Using native drug fluorescence, their distributions in tissue cryosections were related to the underlying tumor vasculature, tumor cell proliferation, and apoptosis. Topotecan exhibited a relatively uniform tumor distribution; in tissue 100 μm away from vessels, it reached 94% ± 5% of levels seen around blood vessels, whereas irinotecan and irinophore C were found to reach only 41% ± 10% and 5% ± 2%, respectively. Surprisingly, all three agents were able to initially inhibit proliferation uniformly throughout the tumors, and it was their rate of washout (topotecan > irinotecan > irinophore C) that correlated with activity. To explain this discrepancy, we looked at SN38, the active metabolite of irinotecan, and found it to penetrate tissue similarly to topotecan. Hence, the poor access to the tumor compartment of irinotecan and irinophore C could be offset by their systemic conversion to SN38. It was concluded that all three agents were effective at reaching tumor cells, and that despite the poor access to the extravascular compartment of irinophore C, its extended plasma exposure and systemic conversion to the diffusible metabolite SN38 enabled it to effectively target solid tumors. Mol Cancer Ther; 13(11); 2727–37. ©2014 AACR.
The PI3K/AKT/mTOR pathway is frequently activated in head and neck squamous cell carcinoma (HNSCC), but pathway inhibition has variable efficacy. Identification of predictive biomarkers and mechanisms of resistance would allow selection of patients most likely to respond and novel therapeutic combinations. The purpose of this study was to extend recent discoveries regarding the PI3K/AKT/mTOR pathway in HNSCC by more broadly examining potential biomarkers of response, by examining pathway inhibitors with a diverse range of targets, and by defining mechanisms of resistance and potential combination therapies. We used reverse-phase protein arrays (RPPA) to simultaneously evaluate expression of 195 proteins; SNP array to estimate gene copy number; and mass array to identify mutations. We examined altered signaling at baseline and after pathway inhibition. Likewise, we examined the activation of the PI3K/AKT/mTOR pathway in HNSCC tumors by RPPA. Cell lines with PIK3CA mutations were sensitive to pathway inhibitors, whereas amplification status did not predict sensitivity. While we identified a set of individual candidate biomarkers of response to pathway inhibitors, proteomic pathway scores did not correlate with amplification or mutation and did not predict response. Several receptor tyrosine kinases, including EGFR and ERK, were activated following PI3K inhibition in resistant cells; dual pathway inhibition of PI3K and EGFR or MEK demonstrated synergy. Combined MEK and PI3K inhibition was markedly synergistic in HRAS-mutant cell lines. Our findings indicate that clinical trials of single-agent PI3K/AKT/mTOR pathway inhibitors in selected populations and of PI3K/EGFR or PI3K/MEK inhibitor combinations are warranted; we plan to conduct such trials. Mol Cancer Ther; 13(11); 2738–50. ©2014 AACR.
A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. While these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug–protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib), and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune, and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes, and signaling pathways that are simultaneously engaged by multitargeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments. Mol Cancer Ther; 13(11); 2751–62. ©2014 AACR.
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