Basal cell carcinoma (BCC) is the most commonly diagnosed cancer. While most BCCs are amenable to surgery, some tumors can reach a more advanced stage or metastasize, and become ineligible for surgical resection or radiotherapy. Abnormal activation of the Hedgehog (Hh) pathway is a key driver in BCC pathophysiology. Consequently, inhibitors of the Hh pathway have been developed. Molecules that inhibit the receptor protein Smoothened (SMO) are the most advanced in clinical development. Vismodegib is the first-in-class SMO inhibitor and has been approved in a number of countries for the treatment of metastatic or locally advanced BCC. Several molecules have demonstrated antitumoral activity, but treatment may be limited in duration by a number of side effects, and it is not yet established whether these agents are truly curative or whether continued treatment will be required. Resistance to SMO inhibition has been reported in the clinic for which incidence and mechanisms must be elucidated to inform future therapeutic strategies. Intermittent dosing regimens to improve tolerability, as well as neoadjuvant use of Hh pathway inhibitors, are currently under investigation. Here, we review the most recent outcomes obtained with Hh inhibitors under clinical investigation in BCC. Mol Cancer Ther; 14(3); 633–41. ©2015 AACR.
Hsp70 is a stress-inducible molecular chaperone that is required for cancer development at several steps. Targeting the active site of Hsp70 has proven relatively challenging, driving interest in alternative approaches. Hsp70 collaborates with the Bcl2-associated athanogene 3 (Bag3) to promote cell survival through multiple pathways, including FoxM1. Therefore, inhibitors of the Hsp70–Bag3 protein–protein interaction (PPI) may provide a noncanonical way to target this chaperone. We report that JG-98, an allosteric inhibitor of this PPI, indeed has antiproliferative activity (EC50 values between 0.3 and 4 μmol/L) across cancer cell lines from multiple origins. JG-98 destabilized FoxM1 and relieved suppression of downstream effectors, including p21 and p27. On the basis of these findings, JG-98 was evaluated in mice for pharmacokinetics, tolerability, and activity in two xenograft models. The results suggested that the Hsp70–Bag3 interaction may be a promising, new target for anticancer therapy. Mol Cancer Ther; 14(3); 642–8. ©2015 AACR.
p53 is a critical tumor suppressor and is the most frequently inactivated gene in human cancer. Inhibition of the interaction of p53 with its negative regulator MDM2 represents a promising clinical strategy to treat p53 wild-type tumors. AMG 232 is a potential best-in-class inhibitor of the MDM2–p53 interaction and is currently in clinical trials. We characterized the activity of AMG 232 and its effect on p53 signaling in several preclinical tumor models. AMG 232 binds the MDM2 protein with picomolar affinity and robustly induces p53 activity, leading to cell-cycle arrest and inhibition of tumor cell proliferation. AMG 232 treatment inhibited the in vivo growth of several tumor xenografts and led to complete and durable regression of MDM2-amplified SJSA-1 tumors via growth arrest and induction of apoptosis. Therapeutic combination studies of AMG 232 with chemotherapies that induce DNA damage and p53 activity resulted in significantly superior antitumor efficacy and regression, and markedly increased activation of p53 signaling in tumors. These preclinical data support the further evaluation of AMG 232 in clinical trials as both a monotherapy and in combination with standard-of-care cytotoxics. Mol Cancer Ther; 14(3); 649–58. ©2015 AACR.
A common hallmark of cancers with highly aggressive phenotypes is increased proteolysis in the tumor and the surrounding microenvironment. Prostate cancer has a number of proteases uniquely associated with it that may play various important roles in disease progression. In this report, we utilize the peritumoral proteolytic activity of prostate cancer to activate engineered peptide constructs for the treatment and noninvasive imaging of prostate cancer. Using a modular "propeptide" approach, a cationic diastereomeric pore-forming peptide domain was linked to an inactivating acidic peptide domain. The inactivating acidic peptide domain was engineered to be a cleavable substrate for the secreted serine protease prostate-specific antigen (PSA) or the transmembrane metalloprotease prostate-specific membrane antigen (PSMA). The propeptides were then evaluated in a direct comparison study. Both the PSA and PSMA activated propeptides were found to be cytotoxic to prostate cancer cells in vitro. In vivo, however, treatment of LNCaP and CWR22Rv1 xenografts with the PSMA propeptide resulted in a pronounced cytostatic effect when compared with xenografts treated with the PSA propeptide or the cationic diastereomeric peptide alone. The PSMA activated propeptide also proved to be an effective optical imaging probe in vivo when labeled with a near-infrared fluorophore. These data suggest that protease-activated pore-forming peptides could potentially be used for both imaging and treating prostate cancer. Mol Cancer Ther; 14(3); 659–68. ©2014 AACR.
HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity. Mol Cancer Ther; 14(3); 669–80. ©2015 AACR.
Primary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for therapeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derangement of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading proteinase (IdeS). Similar to cancer-related matrix metalloproteinases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost antibody-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-suppressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzumab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity. Mol Cancer Ther; 14(3); 681–91. ©2014 AACR.
SYD985 is a HER2-targeting antibody–drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need. Mol Cancer Ther; 14(3); 692–703. ©2015 AACR.
Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3–BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3–BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3–BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3–BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-BAR cells expressing an FGFR3–BAIAP2L1 variant lacking the Bin–Amphiphysin–Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-BAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity. Mol Cancer Ther; 14(3); 704–12. ©2015 AACR.
Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b. Mol Cancer Ther; 14(3); 713–26. ©2014 AACR.
Uncontrolled Hedgehog (Hh) signaling is the cause of several malignancies, including the pediatric cancer medulloblastoma, a neuroectodermal tumor affecting the cerebellum. Despite the development of potent Hh pathway antagonists, medulloblastoma drug resistance is still an unresolved issue that requires the identification of novel drug targets. Following up on our observation that histone deacetylase 6 (HDAC6) expression was increased in Hh-driven medulloblastoma, we found that this enzyme is essential for full Hh pathway activation. Intriguingly, these stimulatory effects of HDAC6 are partly integrated downstream of primary cilia, a known HDAC6-regulated structure. In addition, HDAC6 is also required for the complete repression of basal Hh target gene expression. These contrasting effects are mediated by HDAC6's impact on Gli2 mRNA and GLI3 protein expression. As a result of this complex interaction with Hh signaling, global transcriptome analysis revealed that HDAC6 regulates only a subset of Smoothened- and Gli-driven genes, including all well-established Hh targets such as Ptch1 or Gli1. Importantly, medulloblastoma cell survival was severely compromised by HDAC6 inhibition in vitro and pharmacologic HDAC6 blockade strongly reduced tumor growth in an in vivo allograft model. In summary, our data describe an important role for HDAC6 in regulating the mammalian Hh pathway and encourage further studies focusing on HDAC6 as a novel drug target in medulloblastoma. Mol Cancer Ther; 14(3); 727–39. ©2014 AACR.
CD30 is a cytokine receptor belonging to the TNF superfamily (TNFRSF8) that acts as a regulator of apoptosis. The presence of CD30 antigen is important in the diagnosis of Hodgkin disease and anaplastic large cell lymphoma. There have been sporadic reports of CD30 expression in nonlymphoid tumors, including malignant mesothelioma. Given the remarkable success of brentuximab vedotin, an antibody–drug conjugate directed against CD30 antigen, in lymphoid malignancies, we undertook a study to examine the incidence of CD30 in mesothelioma and to investigate the ability to target CD30 antigen in mesothelioma. Mesothelioma tumor specimens (N = 83) were examined for CD30 expression by IHC. Positive CD30 expression was noted in 13 mesothelioma specimens, primarily those of epithelial histology. There was no significant correlation of CD30 positivity with tumor grade, stage, or survival. Examination of four mesothelioma cell lines (H28, H2052, H2452, and 211H) for CD30 expression by both FACS analysis and confocal microscopy showed that CD30 antigen localized to the cell membrane. Brentuximab vedotin treatment of cultured mesothelioma cells produced a dose-dependent decrease in cell growth and viability at clinically relevant concentrations. Our studies validate the presence of CD30 antigen in a subgroup of epithelial-type mesothelioma tumors and indicate that selected mesothelioma patients may derive benefit from brentuximab vedotin treatment. Mol Cancer Ther; 14(3); 740–6. ©2015 AACR.
Patients with ovarian cancer are generally diagnosed at FIGO (International Federation of Gynecology and Obstetrics) stage III/IV, when ascites is common. The volume of ascites correlates positively with the extent of metastasis and negatively with prognosis. Membrane GRP78, a stress-inducible endoplasmic reticulum chaperone that is also expressed on the plasma membrane (memGRP78) of aggressive cancer cells, plays a crucial role in the embryonic stem cell maintenance. We studied the effects of ascites on ovarian cancer stem-like cells using a syngeneic mouse model. Our study demonstrates that ascites-derived tumor cells from mice injected intraperitoneally with murine ovarian cancer cells (ID8) express increased memGRP78 levels compared with ID8 cells from normal culture. We hypothesized that these ascites-associated memGRP78+ cells are cancer stem-like cells (CSC). Supporting this hypothesis, we show that memGRP78+ cells isolated from murine ascites exhibit increased sphere forming and tumor initiating abilities compared with memGRP78– cells. When the tumor microenvironment is recapitulated by adding ascites fluid to cell culture, ID8 cells express more memGRP78 and increased self-renewing ability compared with those cultured in medium alone. Moreover, compared with their counterparts cultured in normal medium, ID8 cells cultured in ascites, or isolated from ascites, show increased stem cell marker expression. Antibodies directed against the carboxy-terminal domain of GRP78: (i) reduce self-renewing ability of murine and human ovarian cancer cells preincubated with ascites and (ii) suppress a GSK3α-AKT/SNAI1 signaling axis in these cells. Based on these data, we suggest that memGRP78 is a logical therapeutic target for late-stage ovarian cancer. Mol Cancer Ther; 14(3); 747–56. ©2015 AACR.
The FDA-approved BRAF inhibitor vemurafenib achieves outstanding clinical response rates in patients with melanoma, but early resistance is common. Understanding the pathologic mechanisms of drug resistance and identification of effective therapeutic alternatives are key scientific challenges in the melanoma setting. Using proteomic techniques, including shotgun analysis and 2D-gel electrophoresis, we identified a comprehensive signature of the vemurafenib-resistant M24met in comparison with the vemurafenib-sensitive A375 melanoma cell line. The resistant cells were characterized by loss of differentiation, induction of transformation, enhanced expression of the lysosomal compartment, increased potential for metastasis, migration, adherence and Ca2+ ion binding, enhanced expression of the MAPK pathway and extracellular matrix proteins, and epithelial–mesenchymal transformation. The main features were verified by shotgun analysis with QEXACTIVE orbitrap MS, electron microscopy, lysosomal staining, Western blotting, and adherence assay in a VM-1 melanoma cell line with acquired vemurafenib resistance. On the basis of the resistance profile, we were able to successfully predict that a novel resveratrol-derived COX-2 inhibitor, M8, would be active against the vemurafenib-resistant but not the vemurafenib-sensitive melanoma cells. Using high-throughput methods for cell line and drug characterization may thus offer a new way to identify key features of vemurafenib resistance, facilitating the design of effective rational therapeutic alternatives. Mol Cancer Ther; 14(3); 757–68. ©2015 AACR.
Triple-negative breast cancer (TNBC) has the lowest 5-year survival rate of invasive breast carcinomas, and currently there are no approved targeted therapies for this aggressive form of the disease. The androgen receptor (AR) is expressed in up to one third of TNBC and we find that all AR+ TNBC primary tumors tested display nuclear localization of AR, indicative of transcriptionally active receptors. While AR is most abundant in the "luminal AR (LAR)" molecular subtype of TNBC, here, for the first time, we use both the new-generation anti-androgen enzalutamide and AR knockdown to demonstrate that the other non-LAR molecular subtypes of TNBC are critically dependent on AR protein. Indeed, AR inhibition significantly reduces baseline proliferation, anchorage-independent growth, migration, and invasion and increases apoptosis in four TNBC lines (SUM159PT, HCC1806, BT549, and MDA-MB-231), representing three non-LAR TNBC molecular subtypes (mesenchymal-like, mesenchymal stem–like, and basal-like 2). In vivo, enzalutamide significantly decreases viability of SUM159PT and HCC1806 xenografts. Furthermore, mechanistic analysis reveals that AR activation upregulates secretion of the EGFR ligand amphiregulin (AREG), an effect abrogated by enzalutamide in vitro and in vivo. Exogenous AREG partially rescues the effects of AR knockdown on proliferation, migration, and invasion, demonstrating that upregulation of AREG is one mechanism by which AR influences tumorigenicity. Together, our findings indicate that non-LAR subtypes of TNBC are AR dependent and, moreover, that enzalutamide is a promising targeted therapy for multiple molecular subtypes of AR+ TNBC. Mol Cancer Ther; 14(3); 769–78. ©2015 AACR.
Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch–) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch+ cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch+ cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch– cells failed to generate tumors. -Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch+ cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics. Mol Cancer Ther; 14(3); 779–87. ©2015 AACR.
Pancreatic ductal adenocarcinoma (PDAC) frequently develops therapeutic resistances, which can be divided into extrinsic and intrinsic resistance. The extrinsic resistance that arises from the surrounding dense tumor stroma is much better understood. However, the mechanisms of intrinsic resistance are not well understood. Here, we report that reactive oxygen species (ROS) induced by gemcitabine treatment, a newly discovered cytotoxic activity, served as a probe in our study to reveal the mechanisms of the intrinsic therapeutic resistance. Our results showed that gemcitabine-induced ROS is generated by NOX and through the increase of p22–phox expression via NF-B activation. As a feedback mechanism, nuclear translocation of Nrf2 stimulated the transcription of cytoprotective antioxidant genes, especially genes encoding enzymes that catalyze glutathione (GSH) production to reduce elevated ROS as an intrinsic resistance countermeasure. RNAi-mediated depletion of Nrf2 or addition of β-phenylethyl isothiocyanate inhibited the ROS detoxification process by reducing GSH levels, which, in turn, increased the efficacy of gemcitabine in vitro and in vivo. Thus, our study suggests that a redox-mediated pathway contributes to the intrinsic resistance of PDAC to gemcitabine and provides a basis for developing strategies to preferentially kill PDAC cells through ROS-mediated mechanism. The combination of gemcitabine and PEITC has a selective cytotoxic effect against pancreatic cancer cells in vivo and could thus prove valuable as a cancer treatment. Mol Cancer Ther; 14(3); 788–98. ©2014 AACR.
Agents targeting insulin-like growth factor 1 receptor (IGF-1R) are being actively examined in clinical trials. Although there has been some initial success of single-agent targeting IGF-1R, attempts in later studies failed because of resistance. This study aimed to understand the effects of programmed cell death 4 (Pdcd4) on the chemosensitivity of the IGF-1R inhibitor OSI-906 in colorectal cancer cells and the mechanism underlying this impact. Using OSI-906–resistant and –sensitive colorectal cancer cells, we found that the Pdcd4 level directly correlates with cell chemosensitivity to OSI-906. In addition, tumors derived from Pdcd4 knockdown cells resist the growth inhibitory effect of OSI-906 in a colorectal cancer xenograft mouse model. Moreover, Pdcd4 enhances the antiproliferative effect of OSI-906 in resistant cells through suppression of p70S6K1 activation. Knockdown of p70S6K1, but not p70S6K2, significantly increases the chemosensitivity of OSI-906 in cultured colorectal cancer cells. Furthermore, the combination of OSI-906 and PF-4708671, a p70S6K1 inhibitor, efficiently suppresses the growth of OSI-906–resistant colon tumor cells in vitro and in vivo. Taken together, activation of p70S6K1 that is inhibited by Pdcd4 is essential for resistance to the IGF-1R inhibitor in colon tumor cells, and the combinational treatment of OSI-906 and PF-4708671 results in enhanced antiproliferation effects in colorectal cancer cells in vitro and in vivo, providing a novel venue to overcome the resistance to the IGF-1R inhibitor in treating colorectal cancer. Mol Cancer Ther; 14(3); 799–809. ©2015 AACR.
Blockade of EGFR has been proved useful in enhancing the effect of radiotherapy, but the advantages of new-generation EGFR tyrosine kinase inhibitors (TKI) in radiosensitization are not well known. We used two human bladder cancer cells with wild-type EGFR to study the synergism between irradiation and afatinib (an EGFR/HER2 dual kinase inhibitor) or erlotinib (an EGFR kinase inhibitor). Here, we showed that afatinib has better radiosensitizing effect than erlotinib in increasing cancer cell killing, the percentage of apoptotic cells, and DNA damage. Afatinib is also superior to erlotinib in combining radiation to decrease tumor size, inhibit glucose metabolism, and enhance apoptotic proteins in vivo. Finally, erlotinib suppressed cell growth and induced more DNA damage in bladder cancer cells transfected with HER2 shRNA, but not in control vector-treated cells. In conclusion, concomitant blockade of radiation-activated EGFR and HER2 signaling by a new-generation EGFR TKI better inhibits the growth of bladder cancer cells both in vitro and in vivo. The absence of radiosensitization by EGFR inhibition alone and the greater radiosensitizing effect of EGFR inhibitor in HER2 knocked down cells suggest the synergism between HER2 and EGFR in determining radiosensitivity. The regained radiosensitizing activity of erlotinib implies that with proper HER2 inhibition, EGFR tyrosine kinase is still a potential target to enhance radiotherapy effect in these seemingly unresponsive bladder cancer cells. Mol Cancer Ther; 14(3); 810–20. ©2015 AACR.
Circulating tumor cells (CTC) are detected by the CellSearch System in 20% to 25% of patients with primary breast cancer (pBC). To improve CTC detection, we investigated melanoma cell adhesion molecule (MCAM) as enrichment marker next to epithelial cell adhesion molecule (EpCAM) and tested the clinical relevance of MCAM-positive CTCs in patients with HER2-negative stage II/III pBC starting neoadjuvant chemotherapy (NAC) in the NEOZOTAC trial. Using the CellSearch System, EpCAM-positive and MCAM-positive CTCs were separately enriched from 7.5 mL blood, at baseline and after the first NAC cycle. Circulating endothelial cells (CEC) were measured using flow cytometry. Primary objective was to improve the CTC detection rate to ≥40% combining EpCAM/MCAM. Correlations of CTC and CEC counts and pathologic complete response (pCR) were also explored. At baseline, we detected EpCAM-positive and MCAM-positive CTCs in 12 of 68 (18%) and 8 of 68 (12%) patients, respectively. After one cycle, this was 7 of 44 (16%) and 7 of 44 (16%) patients, respectively. The detection rate improved from 18% at baseline and 16% after one cycle with EpCAM to 25% (P = 0.08) and 30% (P = 0.02), respectively, with EpCAM/MCAM. No patients with MCAM-positive CTCs versus 23% of patients without MCAM-positive CTCs at baseline achieved pCR (P = 0.13). EpCAM-positive CTCs and CEC counts were not correlated to pCR. Combined EpCAM/MCAM CellSearch enrichment thus increased the CTC detection rate in stage II/III pBC. We found no associations of CTC and CEC counts with pCR to NAC. The clinical relevance of MCAM-positive CTCs deserves further study. Mol Cancer Ther; 14(3); 821–7. ©2014 AACR.
Despite significant advances in the treatment of head and neck squamous cell carcinoma (HNSCC), the survival rate has not changed in the last decades. Therefore, the development of novel therapeutic strategies is pursued. Cancer–testis antigens (CTA) are strong immunogenic proteins with a tumor-restricted expression pattern, and are considered ideal targets for tumor-specific immunotherapeutic approaches. In this study, using an in silico approach, we selected, among 139 previously described CTA, candidates to be evaluated in 89 HNSCC and 20 normal mucosa samples. SPANX-CD (71.9%), MAGEB2 (44.9%), MAGEA1 (44.9%), MAGEB6 (32.6%), and CXORF48 (27.0%) were found frequently expressed in HNSCC, and over 85% of the tumors expressed at least one of these five CTAs. The mRNA positivity of CXORF48, MAGEB6, and CRISP2 presented significant associations with recognized clinical features for poor outcome. Furthermore, MAGEA3/6 positivity was associated with significantly better disease-free survival (DFS, P = 0.014), and the expression of this antigen was shown to be an independent prognostic factor for tumor recurrence. In conclusion, one of five selected CTAs is expressed in at least 85% of the HNSCCs, suggesting a possible usage as target for immunotherapeutic approaches, and the mRNA-positivity for MAGEA3/6 is shown to be an independent marker for DFS. Mol Cancer Ther; 14(3); 828–34. ©2015 AACR.
Circulating tumor cells (CTC) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of patients with cancer. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared with the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of patients with breast and pancreatic cancer, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types. Mol Cancer Ther; 14(3); 835–43. ©2015 AACR.
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