The European Network for Cancer Research in Children and Adolescents consortium organized a workshop in Rome, in June 2012, on "Biology-Driven Drug Development Renal Tumors Workshop" to discuss the current knowledge in pediatric renal cancers and to recommend directions for further research. Wilms tumor is the most common renal tumor of childhood and represents a success of pediatric oncology, with cure rates of more than 85% of cases. However, a substantial minority (~25%) responds poorly to current therapies and requires "high-risk" treatment or relapse. Moreover, the successfully treated majority are vulnerable to the late effects of treatment, with nearly one quarter reporting severe chronic health conditions by 25 years of follow-up. Main purposes of this meeting were to advance our understanding on the molecular drivers in Wilms tumor, their heterogeneity and interdependencies; to provide updates on the clinical–pathologic associations with biomarkers; to identify eligible populations for targeted drugs; and to model opportunities to use preclinical model systems and prioritize targeted agents for early phase clinical trials. At least three different pathways are involved in Wilms tumor; this review represents the outcome of the workshop discussion on the WNT/β-catenin pathway in Wilms tumorigenesis. Mol Cancer Ther; 12(12); 2619–27. ©2013 AACR.
The urokinase system is overexpressed in epithelial ovarian cancer cells and is expressed at low levels in normal cells. To develop a platform for intracellular and targeted delivery of therapeutics in ovarian cancer, we conjugated urokinase plasminogen activator (uPA) antibodies to liposomal nanobins. The arsenic trioxide–loaded nanobins had favorable physicochemical properties and the ability to bind specifically to uPA. Confocal microscopy showed that the uPA-targeted nanobins were internalized by ovarian cancer cells, whereas both inductively coupled plasma optical mass spectrometry (ICP-MS) and fluorescence-activated cell sorting (FACS) analyses confirmed more than four-fold higher uptake of targeted nanobins when compared with untargeted nanobins. In a coculture assay, the targeted nanobins showed efficient uptake in ovarian cancer cells but not in the normal primary omental mesothelial cells. Moreover, this uptake could be blocked by either downregulating uPA receptor expression in the ovarian cancer cells using short-hairpin RNA (shRNA) or by competition with free uPA or uPA antibody. In proof-of-concept experiments, mice bearing orthotopic ovarian tumors showed a greater reduction in tumor burden when treated with targeted nanobins than with untargeted nanobins (47% vs. 27%; P < 0.001). The targeted nanobins more effectively inhibited tumor cell growth both in vitro and in vivo compared with untargeted nanobins, inducing caspase-mediated apoptosis and impairing stem cell marker, aldehyde dehydrogenase-1A1 (ALDH1A1), expression. Ex vivo fluorescence imaging of tumors and organs corroborated these results, showing preferential localization of the targeted nanobins to the tumor. These findings suggest that uPA-targeted nanobins capable of specifically and efficiently delivering payloads to cancer cells could serve as the foundation for a new targeted cancer therapy using protease receptors. Mol Cancer Ther; 12(12); 2628–39. ©2013 AACR.
Apigenin is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. Apigenin treatment resulted in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between apigenin and ABT-263 in colon cancer cells. We observed a synergistic effect between apigenin and ABT-263 on apoptosis of colon cancer cells. ABT-263 alone induced limited cell death while upregulating expression of Mcl-1, a potential mechanism for the acquired resistance to ABT-263. The presence of apigenin antagonized ABT-263–induced Mcl-1 upregulation and dramatically enhanced ABT-263–induced cell death. Meanwhile, apigenin suppressed AKT and ERK activation. Inactivation of either AKT or ERK by lentivirus-transduced shRNA or treatment with specific small-molecule inhibitors of these pathways enhanced ABT-263–induced cell death, mirroring the effect of apigenin. Moreover, the combination response was associated with upregulation of Bim and activation of Bax. Downregulation of Bax eliminated the synergistic effect of apigenin and ABT-263 on cell death. Xenograft studies in SCID mice showed that the combined treatment with apigenin and ABT-263 inhibited tumor growth by up to 70% without obvious adverse effects, while either agent only inhibited around 30%. Our results demonstrate a novel strategy to enhance ABT-263–induced antitumor activity in human colon cancer cells by apigenin via inhibition of the Mcl-1, AKT, and ERK prosurvival regulators. Mol Cancer Ther; 12(12); 2640–50. ©2013 AACR.
Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine–based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy. Mol Cancer Ther; 12(12); 2651–62. ©2013 AACR.
Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma. Mol Cancer Ther; 12(12); 2663–74. ©2013 AACR.
Inhibition of Wee1 is emerging as a novel therapeutic strategy for cancer, and some data suggest that cells with dysfunctional p53 are more sensitive to Wee1 inhibition combined with conventional chemotherapy than those with functional p53. We and others found that Wee1 inhibition sensitizes leukemia cells to cytarabine. Thus, we sought to determine whether chemosensitization by Wee1 inhibition is dependent on p53 dysfunction and whether combining Wee1 inhibition is tolerable and effective in vivo. Synergistic inhibition of proliferation with a Wee1 inhibitor in clinical development, MK1775, and cytarabine was observed in all acute myelogenous leukemia (AML) cell lines tested, regardless of p53 functionality. Mechanistic studies indicate that inhibition of Wee1 abrogates the S-phase checkpoint and augments apoptosis induced by cytarabine. In AML and lung cancer cell lines, genetic disruption of p53 did not alter the cells' enhanced sensitivity to antimetabolites with Wee1 inhibition. Finally, mice with AML were treated with cytarabine and/or MK1775. The combination of MK1775 and cytarabine was well tolerated in mice and enhanced the antileukemia effects of cytarabine, including survival. Thus, inhibition of Wee1 sensitizes hematologic and solid tumor cell lines to antimetabolite chemotherapeutics, whether p53 is functional or not, suggesting that the use of p53 mutation as a predictive biomarker for response to Wee1 inhibition may be restricted to certain cancers and/or chemotherapeutics. These data provide preclinical justification for testing MK1775 and cytarabine in patients with leukemia. Mol Cancer Ther; 12(12); 2675–84. ©2013 AACR.
VEGF receptor (VEGFR) signaling plays a key role in tumor angiogenesis. Although some VEGFR signal-targeted drugs have been approved for clinical use, their utility is limited by associated toxicities or resistance to such therapy. To overcome these limitations, we developed TAS-115, a novel VEGFR and hepatocyte growth factor receptor (MET)-targeted kinase inhibitor with an improved safety profile. TAS-115 inhibited the kinase activity of both VEGFR2 and MET and their signal-dependent cell growth as strongly as other known VEGFR or MET inhibitors. On the other hand, kinase selectivity of TAS-115 was more specific than that of sunitinib and TAS-115 produced relatively weak inhibition of growth (GI50 > 10 μmol/L) in VEGFR signal- or MET signal-independent cells. Furthermore, TAS-115 induced less damage in various normal cells than did other VEGFR inhibitors. These data suggest that TAS-115 is extremely selective and specific, at least in vitro. In in vivo studies, TAS-115 completely suppressed the progression of MET-inactivated tumor by blocking angiogenesis without toxicity when given every day for 6 weeks, even at a serum-saturating dose of TAS-115. The marked selectivity of TAS-115 for kinases and targeted cells was associated with improved tolerability and contributed to the ability to sustain treatment without dose reduction or a washout period. Furthermore, TAS-115 induced marked tumor shrinkage and prolonged survival in MET-amplified human cancer–bearing mice. These data suggest that TAS-115 is a unique VEGFR/MET-targeted inhibitor with improved antitumor efficacy and decreased toxicity. Mol Cancer Ther; 12(12); 2685–96. ©2013 AACR.
Cancers of the urinary bladder result in aggressive and highly angiogenic tumors for which standard treatments have only limited success. Patients with advanced disease have a 5-year survival rate of less than 20%, and no new anticancer agent has been successfully introduced into the clinic armamentarium for the treatment of bladder cancer in more than 20 years. Investigations have identified plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, as being highly expressed in several malignancies, including bladder cancer, in which high expression is associated with a poor prognosis. In this study, we evaluated PAI-1 as a potential therapeutic target for bladder cancer. PAI-1 expression was manipulated in a panel of cell lines and functional inhibition was achieved using the small molecule tiplaxtinin. Reduction or inhibition of PAI-1 resulted in the reduction of cellular proliferation, cell adhesion, and colony formation, and the induction of apoptosis and anoikis in vitro. Treatment of T24 xenografts with tiplaxtinin resulted in inhibition of angiogenesis and induction of apoptosis, leading to a significant reduction in tumor growth. Similar results were obtained through evaluation of the human cervical cancer HeLa cell line, showing that PAI-1–mediated effects are not restricted to tumor cells of bladder origin. Collectively, these data show that targeting PAI-1 may be beneficial and support the notion that novel drugs such as tiplaxtinin could be investigated as anticancer agents. Mol Cancer Ther; 12(12); 2697–708. ©2013 AACR.
Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited. Mol Cancer Ther; 12(12); 2709–21. ©2013 AACR.
Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb–Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 μmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb–Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb–Raf-1 interaction might be a promising strategy targeting pancreatic cancer. Mol Cancer Ther; 12(12); 2722–34. ©2013 AACR.
Cancer cells can be specifically driven into apoptosis by activating Death-receptor-4 (DR4; TRAIL-R1) and/or Death-receptor-5 (DR5; TRAIL-R2). Albeit showing promising preclinical efficacy, first-generation protein therapeutics addressing this pathway, especially agonistic anti-DR4/DR5-monoclonal antibodies, have not been clinically successful to date. Due to their bivalent binding mode, effective apoptosis induction by agonistic TRAIL-R antibodies is achieved only upon additional events leading to antibody-multimer formation. The binding of these multimers to their target subsequently leads to effective receptor-clustering on cancer cells. The research results presented here report on a new class of TRAIL-receptor agonists overcoming this intrinsic limitation observed for antibodies in general. The main feature of these agonists is a TRAIL-mimic consisting of three TRAIL-protomer subsequences combined in one polypeptide chain, termed the single-chain TRAIL-receptor–binding domain (scTRAIL-RBD). In the active compounds, two scTRAIL-RBDs with three receptor binding sites each are brought molecularly in close proximity resulting in a fusion protein with a hexavalent binding mode. In the case of APG350—the prototype of this engineering concept—this is achieved by fusing the Fc-part of a human immunoglobulin G1 (IgG1)-mutein C-terminally to the scTRAIL–RBD polypeptide, thereby creating six receptor binding sites per drug molecule. In vitro, APG350 is a potent inducer of apoptosis on human tumor cell lines and primary tumor cells. In vivo, treatment of mice bearing Colo205-xenograft tumors with APG350 showed a dose-dependent antitumor efficacy. By dedicated muteins, we confirmed that the observed in vivo efficacy of the hexavalent scTRAIL–RBD fusion proteins is—in contrast to agonistic antibodies—independent of FcR-based cross-linking events. Mol Cancer Ther; 12(12); 2735–47. ©2013 AACR.
Heterodimeric Fc designed by engineering the CH3 homodimeric interface of immunoglobulin G1 serves as an attractive scaffold for the generation of bispecific antibodies (bsAb) due to the favorable properties of the Fc region. In this study, we describe a heterodimeric Fc generated by substituting the conserved electrostatic interactions at the CH3 core interface with asymmetric hydrophobic interactions and introducing asymmetric, long-range electrostatic interactions at the rim of the CH3 interface. Coexpression of Fc proteins carrying the combined CH3 variant pairs in HEK293F cells produced the heterodimer, which was purified with more than 90% yield, and retained wild-type Fc biophysical properties. The heterodimeric Fc was exploited to generate a bsAb simultaneously targeting both the Met receptor tyrosine kinase and the VEGF receptor 2 (VEGFR-2), with two respective antigen-specific, single-chain variable fragments (scFv) into the N-terminus. The Met x VEGFR-2 bsAb bound concurrently to the two target antigens, efficiently inhibited the downstream signaling and tube formation stimulated by the two receptors in human endothelial cells, and exhibited more potent antitumor efficacy in MKN45 human gastric cancer xenograft models than both the parent monospecific antibody alone. Collectively, based on the newly designed heterodimeric Fc-based bsAb, our results provide the therapeutic potential of bsAb targeting both Met and VEGFR-2 simultaneously for the treatment of human cancers. Mol Cancer Ther; 12(12); 2748–59. ©2013 AACR.
During melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an epithelial–mesenchymal transition (EMT) process associated with the acquisition of an invasive, prometastatic phenotype. The fibroblast growth factor-2 (FGF2)/FGF receptor (FGFR) system plays a pivotal role in melanoma, leading to autocrine/paracrine induction of tumor cell proliferation and angiogenesis. Long pentraxin-3 (PTX3) interacts with FGF2, and other FGF family members, inhibiting FGF-dependent neovascularization and tumor growth. Here, PTX3 protein and the PTX3-derived acetylated pentapeptide Ac-ARPCA-NH2 inhibit FGF2-driven proliferation and downstream FGFR signaling in murine melanoma B16-F10 cells. Moreover, human PTX3-overexpressing hPTX_B16-F10 cells are characterized by the reversed transition from a mesenchymal to an epithelial-like appearance, inhibition of cell proliferation, loss of clonogenic potential, reduced motility and invasive capacity, downregulation of various mesenchymal markers, and upregulation of the epithelial marker E-cadherin. Accordingly, PTX3 affects cell proliferation and EMT transition in human A375 and A2058 melanoma cells. Also, hPTX_B16-F10 cells showed a reduced tumorigenic and metastatic activity in syngeneic C57BL/6 mice. In conclusion, PTX3 inhibits FGF/FGFR-driven EMT in melanoma cells, hampering their tumorigenic and metastatic potential. These data represent the first experimental evidence about a nonredundant role of the FGF/FGFR system in the modulation of the EMT process in melanoma and indicate that PTX3 or its derivatives may represent the basis for the design of novel therapeutic approaches in FGF/FGFR-dependent tumors, including melanoma. Mol Cancer Ther; 12(12); 2760–71. ©2013 AACR.
Sym004 represents a novel EGF receptor (EGFR)-targeting approach comprising a mixture of two anti-EGFR antibodies directed against distinct epitopes of EGFR. In contrast with single anti-EGFR antibodies, Sym004 induces rapid and highly efficient degradation of EGFR. In the current study, we examine the capacity of Sym004 to augment radiation response in lung cancer and head and neck cancer model systems. We first examined the antiproliferative effect of Sym004 and confirmed 40% to 60% growth inhibition by Sym004. Using clonogenic survival analysis, we identified that Sym004 potently increased cell kill by up to 10-fold following radiation exposure. A significant increase of H2AX foci resulting from DNA double-strand breaks was observed in Sym004-treated cells following exposure to radiation. Mechanistic studies further showed that Sym004 enhanced radiation response via induction of cell-cycle arrest followed by induction of apoptosis and cell death, reflecting inhibitory effects on DNA damage repair. The expression of several critical molecules involved in radiation-induced DNA damage repair was significantly inhibited by Sym004, including DNAPK, NBS1, RAD50, and BRCA1. Using single and fractionated radiation in human tumor xenograft models, we confirmed that the combination of Sym004 and radiation resulted in significant tumor regrowth delay and superior antitumor effects compared with treatment with Sym004 or radiation alone. Taken together, these data reveal the strong capacity of Sym004 to augment radiation response in lung and head and neck cancers. The unique action mechanism of Sym004 warrants further investigation as a promising EGFR targeting agent combined with radiotherapy in cancer therapy. Mol Cancer Ther; 12(12); 2772–81. ©2013 AACR.
Combination chemotherapy is standard for metastatic colorectal cancer; however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic colorectal cancer, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in colorectal cancer compared with its levels in normal mucosa and in chemoresistant colorectal cancer cells compared with isogenic chemo-naïve colorectal cancer cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve colorectal cancer cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve colorectal cancer cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in resensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully resensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance. Mol Cancer Ther; 12(12); 2782–91. ©2013 AACR.
Pancreatic cancer cell lines with mutated ras underwent an alternative form of cell death (aponecrosis) when treated concomitantly with clinically achievable concentrations of arsenic trioxide, ascorbic acid, and disulfiram (Antabuse; AAA). AAA's major effects are mediated through generation of intracellular reactive oxygen species (ROS) and more than 50% decline in intracellular ATP. N-acetyl cysteine and a superoxide dismutase mimetic prevented aponecrosis and restored intracellular ATP levels. DIDS (4,4'-diisothiocyanatostilbene-2, 2' disulfonic acid), the pan- Voltage-Dependent Anion Channel (VDAC), -1, 2, 3 inhibitor and short hairpin RNA (shRNA) to VDAC-1 blocked cell death and ROS accumulation. In vivo exposure of AAA led to a 62% reduction in mean tumor size and eliminated tumors in 30% of nude mice with PANC-1 xenografts. We concluded that early caspase-independent apoptosis was shifted to VDAC-mediated "targeted" aponecrosis by the addition of disulfiram to arsenic trioxide and ascorbic acid. Conceptually, this work represents a paradigm shift where switching from apoptosis to aponecrosis death pathways, also known as targeted aponecrosis, could be utilized to selectively kill pancreatic cancer cells resistant to apoptosis. Mol Cancer Ther; 12(12); 2792–803. ©2013 AACR.
We previously showed that in innately resistant tumors, silencing of the estrogen receptor (ER) could be reversed by treatment with a histone deacetylase (HDAC) inhibitor, entinostat. Tumors were then responsive to aromatase inhibitor (AI) letrozole. Here, we investigated whether ER in the acquired letrozole-resistant tumors could be restored with entinostat. Ovariectomized athymic mice were inoculated with MCF-7Ca cells, supplemented with androstenedione (4A), the aromatizable substrate. When the tumors reached about 300 mm3, the mice were treated with letrozole. After initial response to letrozole, the tumors eventually became resistant (doubled their initial volume). The mice then were grouped to receive letrozole, exemestane (250 μg/d), entinostat (50 μg/d), or the combination of entinostat with letrozole or exemestane for 26 weeks. The growth rates of tumors of mice treated with the combination of entinostat with letrozole or exemestane were significantly slower than with the single agent (P < 0.05). Analysis of the letrozole-resistant tumors showed entinostat increased ERα expression and aromatase activity but downregulated Her-2, p-Her-2, p-MAPK, and p-Akt. However, the mechanism of action of entinostat in reversing acquired resistance did not involve epigenetic silencing but rather included posttranslational as well as transcriptional modulation of Her-2. Entinostat treatment reduced the association of the Her-2 protein with HSP-90, possibly by reducing the stability of Her-2 protein. In addition, entinostat also reduced Her-2 mRNA levels and its stability. Our results suggest that the HDAC inhibitor may reverse letrozole resistance in cells and tumors by modulating Her-2 expression and activity. Mol Cancer Ther; 12(12); 2804–16. ©2013 AACR.
Uveal melanoma is the most common primary intraocular malignant tumor in adults and half of the primary tumors will develop fatal metastatic disease to the liver and the lung. Crizotinib, an inhibitor of c-Met, anaplastic lymphoma kinase (ALK), and ROS1, inhibited the phosphorylation of the c-Met receptor but not of ALK or ROS1 in uveal melanoma cells and tumor tissue. Consequently, migration of uveal melanoma cells was suppressed in vitro at a concentration associated with the specific inhibition of c-Met phosphorylation. This effect on cell migration could be recapitulated with siRNA specific to c-Met but not to ALK or ROS1. Therefore, we developed a uveal melanoma metastatic mouse model with EGFP–luciferase-labeled uveal melanoma cells transplanted by retro-orbital injections to test the effect of crizotinib on metastasis. In this model, there was development of melanoma within the eye and also metastases to the liver and lung at 7 weeks after the initial transplantation. When mice were treated with crizotinib starting 1 week after the transplantation, we observed a significant reduction in the development of metastases as compared with untreated control sets. These results indicate that the inhibition of c-Met activity alone may be sufficient to strongly inhibit metastasis of uveal melanoma from forming, suggesting crizotinib as a potential adjuvant therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease. Mol Cancer Ther; 12(12); 2817–26. ©2013 AACR.
Current strategies in cancer treatment employ combinations of different treatment modalities, which include chemotherapy, radiotherapy, immunotherapy, and surgery. Consistent with that approach, the present study demonstrates how chemotherapeutic agents can potentiate the delivery of radiolabeled, necrosis-targeting antibodies (chTNT-3, NHS76) to tumor. All chemotherapeutics in this study (5-fluorouracil, etoposide, vinblastine, paclitaxel, and doxorubicin) resulted in statistically significant increases in tumor uptake of radiolabeled antibodies and their F(ab')2 fragments compared to no pretreatment with chemotherapy. Labeled antibodies were administered at various time points following a single dose of chemotherapy in multiple tumor models, and the biodistribution of the antibodies was determined by measuring radioactivity in harvested tissues. MicroPET/CT was also done to demonstrate clinical relevancy of using chemotherapy pretreatment to increase antibody uptake. Results of biodistribution and imaging data reveal specific time frames following chemotherapy when necrosis-targeting antibodies are best delivered, either for imaging or radiotherapy. Thus, the present work offers the prospect of using cytoreductive chemotherapy to increase tumor accumulation of select therapeutic antibodies, especially when combined with other forms of immunotherapy, for the successful treatment of solid tumors. Mol Cancer Ther; 12(12); 2827–36. ©2013 AACR.
Drugs that target microtubules are potent inhibitors of angiogenesis, but their mechanism of action is not well understood. To explore this, we treated human umbilical vein endothelial cells with paclitaxel, vinblastine, and colchicine and measured the effects on microtubule dynamics and cell motility. In general, lower drug concentrations suppressed microtubule dynamics and inhibited cell migration whereas higher concentrations were needed to inhibit cell division; however, surprisingly, large drug-dependent differences were seen in the relative concentrations needed to inhibit these two processes. Suppression of microtubule dynamics did not significantly affect excursions of lamellipodia away from the nucleus or prevent cells from elongating; but, it did inhibit retraction of the trailing edges that are normally enriched in dynamic microtubules, thereby limiting cell locomotion. Complete removal of microtubules with a high vinblastine concentration caused a loss of polarity that resulted in roundish, rather than elongated, cells, rapid but nondirectional membrane activity, and little cell movement. The results are consistent with a model in which more static microtubules stabilize the leading edge of migrating cells, whereas more dynamic microtubules locate to the rear where they can remodel and allow tail retraction. Suppressing microtubule dynamics interferes with tail retraction, but removal of microtubules destroys the asymmetry needed for cell elongation and directional motility. The prediction that suppressing microtubule dynamics might be sufficient to prevent angiogenesis was supported by showing that low concentrations of paclitaxel could prevent the formation of capillary-like structures in an in vitro tube formation assay. Mol Cancer Ther; 12(12); 2837–46. ©2013 AACR.
Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K-targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin. Mol Cancer Ther; 12(12); 2847–56. ©2013 AACR.
Target-matched treatment with PI3K/AKT/mTOR pathway inhibitors in patients with diverse advanced cancers with PIK3CA mutations have shown promise. Tumors from patients with colorectal cancer were analyzed for PIK3CA, KRAS, and BRAF mutations. PIK3CA-mutated tumors were treated, whenever feasible, with agents targeting the PI3K/AKT/mTOR pathway. Of 194 patients analyzed, 31 (16%) had PIK3CA mutations and 189 (97%) were assessed for KRAS mutations. Patients with PIK3CA mutations had a higher prevalence of simultaneous KRAS mutations than patients with wild-type PIK3CA (71%, 22/31 vs. 43%, 68/158; P = 0.006). Of 31 patients with PIK3CA mutations, 17 (55%) were treated with protocols containing PI3K/AKT/mTOR pathway inhibitors [median age, 57 years; median number of prior therapies, 4; mTORC1 inhibitors (11), phosphoinositide 3-kinase (PI3K) inhibitors (5), or an AKT inhibitor (1)]. None (0/17) had a partial or complete response (PR/CR) and only 1 [6%, 95% confidence interval (CI), 0.01–0.27] had stable disease 6 months or more, which was not significantly different from a stable disease ≥6 month/PR/CR rate of 16% (11/67; 95% CI, 0.09–0.27) in patients with colorectal cancer without PIK3CA mutations treated with PI3K/AKT/mTOR pathway inhibitors (P = 0.44). Median progression-free survival was 1.9 months (95% CI, 1.5–2.3). In conclusion, our data provide preliminary evidence that in heavily pretreated patients with PIK3CA-mutant advanced colorectal cancer, protocols incorporating PI3K/AKT/mTOR inhibitors have minimal activity. PIK3CA mutations are associated with simultaneous KRAS mutations, possibly accounting for therapeutic resistance. Mol Cancer Ther; 12(12); 2857–63. ©2013 AACR.
Insulin-like growth factor (IGF)-binding protein-2 (IGFBP2) expression is increased in various types of cancers, including in a subset of patients with lung cancer. Because IGFBP2 is involved in signal transduction of some critical cancer-related pathways, we analyzed the association between IGFBP2 and response to pathway-targeted agents in seven human non–small cell lung cancer (NSCLC) cell lines. Western blot analysis and ELISA showed that four of the seven NSCLC cell lines analyzed expressed high levels of IGFBP2, whereas the remaining three had barely detectable IGFBP2. Susceptibilities of those seven cell lines to nine anticancer agents targeting to IGF1R, Src, FAK, MEK, and AKT were determined by a dose-dependent cell viability assay. The results showed that high IGFBP2 levels were associated with resistance to dasatinib and, to a lesser degree, to sacaratinib, but not to other agents. Ectopic IGFBP2 overexpression or knockdown revealed that changing IGFBP2 expression levels reversed dasatinib susceptibility phenotype, suggesting a causal relationship between IGFBP2 expression and dasatinib resistance. Molecular characterization revealed that focal adhesion kinase (FAK) activation was associated with increased IGFBP2 expression and partially contributed to IGFBP2-mediated dasatinib resistance. Treatment with a combination of dasatinib and FAK inhibitor led to enhanced antitumor activity in IGFBP2-overexpressing and dasatinib-resistant NSCLC cells in vitro and in vivo. Our results showed that the IGFBP2/FAK pathway is causally associated with dasatinib resistance and may be used as biomarkers for identification of dasatinib responders among patients with lung cancer. Simultaneous targeting on Src and FAK will likely improve the therapeutic efficacy of dasatinib for treatment of lung cancer. Mol Cancer Ther; 12(12); 2864–73. ©2013 AACR.
Hepatocellular carcinoma is highly chemoresistant, and ATP-binding cassette subfamily G member 2 (ABCG2) is thought to play a critical role in this drug resistance. The present study aims to develop effective therapeutic strategies to decrease ABCG2 expression level and to surmount drug resistance in hepatocellular carcinoma chemotherapy. First, we verified a positive correlation between the ABCG2 protein level and the drug resistance of hepatocellular carcinoma cell lines. ABCG2 was preferentially expressed in highly chemoresistant hepatocellular carcinoma cancer stem cells (CSC) enriched with CD133. In addition, ABCG2 was N-linked glycosylated in hepatocellular carcinoma cells, and this modification was involved in sustaining its protein stability. The N-linked glycosylation (NLG) inhibitor tunicamycin dramatically reduced ABCG2 expression, altered its subcellular localization, and reversed its drug efflux effect in multiple hepatocellular carcinoma cell lines. Furthermore, tunicamycin reduced the expression levels of several CSC markers and suppressed the tumorigenicity of CD133+ CSCs. Tunicamycin combined with cisplatin (CDDP) inhibited proliferating cell nuclear antigen (PCNA) expression and increased the cleavage of PARP; this effect was partially rescued by the overexpression of ABCG2 or Akt-myr. The combination therapy more effectively suppressed tumor growth in xenograft mice than did single-agent therapy with either drug. Finally, the CDDP treatment combined with UDP-GlcNAc-dolichol-phosphate N-acetylglucosamine-1 phosphate transferase (DPAGT1) knockdown recapitulated the effect observed when CDDP was used in combination with tunicamycin. In summary, our results suggest that tunicamycin may reverse the drug resistance and improve the efficacy of combination treatments for hepatocellular carcinomas by targeting the DPAGT1/Akt/ABCG2 pathway. Mol Cancer Ther; 12(12); 2874–84. ©2013 AACR.
STAT3 is an important transcriptional factor for cell growth, differentiation, and apoptosis. Although evidence suggests a positive role for STAT3 in cancer, the inhibitory effects of tumor STAT3 on natural killer (NK) cell functions in human hepatocellular carcinoma are unclear. In this study, we found that blocking STAT3 in hepatocellular carcinoma cells enhanced NK-cell antitumor function. In the case of STAT3-blocked hepatocellular carcinoma cells, NKG2D ligands were upregulated, which promoted recognition by NK cells. Importantly, the cytokine profile of hepatocellular carcinoma cells was altered; in particular, TGF-β and interleukin 10 (IL-10) expression was reduced, and type I interferon (IFN) was induced, thus facilitating NK-cell activation. Indeed, the cytotoxicity of NK cells treated with supernatant from STAT3-blocked hepatocellular carcinoma cells was augmented, with a concomitant elevation of molecules associated with NK cytolysis. Further experiments confirmed that the recovery of NK cells depended on the downregulation of TGF-β and upregulation of type I IFN derived from STAT3-blocked hepatocellular carcinoma cells. These findings demonstrated a pivotal role for STAT3 in hepatocellular carcinoma-mediated NK-cell dysfunction, and highlighted the importance of STAT3 blockade for hepatocellular carcinoma immunotherapy, which could restore NK-cell cytotoxicity in addition to its direct influence on tumor cells. Mol Cancer Ther; 12(12); 2885–96. ©2013 AACR.
The EGF receptor (EGFR) is therapeutically targeted by antibodies and small molecules in solid tumors including lung, colorectal, and breast cancer. However, chemotherapy remains important, and efforts to improve efficacy through combination with targeted agents is challenging. This study examined the effects of short and long durations of exposure to the EGFR- and HER2-targeted tyrosine kinase inhibitors (TKI) gefitinib and lapatinib, on induction of cell death and DNA damage by topoisomerase IIα (Topo IIα) poisons, in the SK-Br-3 HER2-amplified breast cancer cell line. Short exposure to either gefitinib or lapatinib for 1 hour did not affect the induction of apoptosis by the Topo IIα poisons doxorubicin, etoposide, and m-AMSA. In contrast, cells treated for 48 hours were resistant to all three drugs. Short exposure (1 hour) to TKI did not alter the number of DNA single- or double-strand breaks (DSB) induced, whereas longer exposure (48 hours) reduced the number of DNA DSBs and the formation of -H2AX foci. Both gefitinib and lapatinib reduced the expression and activity of Topo IIα at 48 hours. Studies using a cell line with inducible downregulation of Topo IIα showed that expression of Topo IIα, and not Topo IIβ, determined the number of DNA strand breaks induced by these chemotherapeutic agents. These results indicate that prolonged exposure to TKIs targeting EGFR and HER2 induce resistance to doxorubicin, etoposide, and m-AMSA through downregulation of Topo IIα. This may explain why their addition to chemotherapy regimens have not increased efficacy. Mol Cancer Ther; 12(12); 2897–908. ©2013 AACR.
Ovarian cancer mortality ranks highest among all gynecologic cancers with growth factor pathways playing an integral role in tumorigenesis, metastatic dissemination, and therapeutic resistance. The HER and VEGF receptor (VEGFR) are both overexpressed and/or aberrantly activated in subsets of ovarian tumors. While agents targeting either the HER or VEGF pathways alone have been investigated, the impact of these agents have not led to overall survival benefit in ovarian cancer. We tested the hypothesis that cotargeting HER and VEGFR would maximize antitumor efficacy at tolerable doses. To this end, ovarian cancer xenografts grown intraperitoneally in athymic nude mice were tested in response to AC480 (pan-HER inhibitor, "HERi"), cediranib (pan-VEGFR inhibitor "VEGFRi"), or BMS-690514 (combined HER/VEGFR inhibitor "EVRi"). EVRi was superior to both HERi and VEGFRi in terms of tumor growth, final tumor weight, and progression-free survival. Correlative tumor studies employing phosphoproteomic antibody arrays revealed distinct agent-specific alterations, with EVRi inducing the greatest overall effect on growth factor signaling. These data suggest that simultaneous inhibition of HER and VEGFR may benefit select subsets of ovarian cancer tumors. To this end, we derived a novel HER/VEGF signature that correlated with poor overall survival in high-grade, late stage, serous ovarian cancer patient tumors. Mol Cancer Ther; 12(12); 2909–16. ©2013 AACR.
Earlier in vitro work demonstrated that PARP inhibition induces cell death in PTEN-null endometrial cancer cell lines, but the in vivo therapeutic efficacy of these agents against endometrial cancer remains unknown. Here, we test the efficacy of AZD2281 (olaparib), an oral PARP inhibitor, in the therapy of PTEN-null endometrial tumors in a preclinical endometrial cancer mouse model. Primary endometrial tumors were generated by epithelial loss of PTEN using an in vivo model. This model recapitulates epithelial-specific loss of PTEN seen in human tumors, and histologically resembles endometrioid carcinomas, the predominant subtype of human endometrial cancers. Olaparib was administered orally to tumor-bearing mice in two hormonal extremes: high or low estrogen. Olaparib treatment achieved a significant reduction in tumor size in a low estrogenic milieu. In striking contrast, no response to olaparib was seen in tumors exposed to high levels of estrogen. Two key observations were made when estrogen levels were dropped: (i) the serum concentration of olaparib was significantly increased, resulting in sustained PARP inhibition at the tumor bed; and (ii) the homologous recombination pathway was compromised, as evidenced by decreased Rad51 protein expression and function. These two mechanisms may account for the sensitization of PTEN-null tumors to olaparib with estrogen deprivation. Results of this preclinical trial suggest that orally administered PARP inhibitors in a low estrogenic hormonal milieu can effectively target PTEN-null endometrial tumors. Extension of this work to clinical trials could personalize the therapy of women afflicted with advanced endometrial cancer using well-tolerated orally administered therapeutic agents. Mol Cancer Ther; 12(12); 2917–28. ©2013 AACR.
Figitumumab (CP-751,871), a potent and fully human monoclonal anti–insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors. Mol Cancer Ther; 12(12); 2929–39. ©2013 AACR.
As acute myelogenous leukemia (AML) patient response to cytarabine-based standard-of-care treatment is variable, stratification into subgroups by biomarker-predicted response may lead to improved clinical outcomes. Here, we assess cell mitochondrial depolarization to proapoptotic signaling BH3-only peptides as a surrogate for the function of Bcl-2 family proteins to address clinical response to cytarabine-based therapy in patients with AML (N = 62). Peripheral blood mononuclear cell (PBMC) or bone marrow aspirate specimens were obtained from newly diagnosed patients with AML, viably preserved, and assayed by flow cytometry following BH3 profile assay with individual BH3 peptides. Mann–Whitney analysis indicates biomarker correlation with response to induction therapy: Notably, BIM priming was highly significant (P = 2 x 10–6) with a compelling sensitivity/specificity profile [area under curve (AUC) = 0.83; 95% confidence interval (CI), 0.73–0.94; P = 2 x 10–10]. Multivariate analysis indicates improved profiles for BIM readout + patient age (AUC = 0.89; 95% CI, 0.81–0.97) and BIM + patient age + cytogenetic status (AUC = 0.91; 95% CI, 0.83–0.98). When patients were stratified by cytogenetic status, BIM readout was significant for both intermediate (P = 0.0017; AUC = 0.88; 95% CI, 0.71–1.04) and unfavorable (P = 0.023; AUC = 0.79; 95% CI, 0.58–1.00) risk groups, demonstrating predictive power independent of cytogenetics. Additional analyses of secondary clinical endpoints displayed correlation between overall survival (P = 0.037) and event-free survival (P = 0.044) when patients were stratified into tertiles by BIM peptide response. Taken together, these results highlight the potential utility of BH3 profiling in personalized diagnostics of AML by offering actionable information for patient management decisions. Mol Cancer Ther; 12(12); 2940–9. ©2013 AACR.
A critical step toward defining tailored therapy in patients with cancer is the identification of genetic interactions that may impair—or boost—the efficacy of selected therapeutic approaches. Cell models able to recapitulate combinations of genetic aberrations are important to find drug–genotype interactions poorly affected by the heterogeneous genetics of human tumors. In order to identify novel pharmacogenomic relationships, we employed an isogenic cell panel that reconstructs cancer genetic scenarios. We screened a library of 43 compounds in human hTERT-HME1 epithelial cells in which PTEN or RB1 were silenced in combination with the targeted knockin of cancer-associated mutations in EGFR, KRAS, BRAF, or PIK3CA oncogenes. Statistical analysis and clustering algorithms were applied to display similar drug response profiles and mutation-specific patterns of activity. From the screen, we discovered that proteasome inhibitors show selectivity toward BRAF V600E–mutant cells, irrespective of PTEN or RB1 expression. Preferential targeting of BRAF-mutant cells by proteasome inhibitors was corroborated in a second BRAF V600E isogenic model, as well as in a panel of colorectal cancer cell lines by the use of the proteasome inhibitor carfilzomib. Notably, carfilzomib also showed striking in vivo activity in a BRAF-mutant human colorectal cancer xenograft model. Vulnerability to proteasome inhibitors is dependent on persistent BRAF signaling, because BRAF V600E blockade by PLX4720 reversed sensitivity to carfilzomib in BRAF-mutant colorectal cancer cells. Our findings indicated that proteasome inhibition might represent a valuable targeting strategy in BRAF V600E–mutant colorectal tumors. Mol Cancer Ther; 12(12); 2950–61. ©2013 AACR.
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