Identification of a Novel NRG1 Fusion with Targeted Therapeutic Implications in Locally Advanced Pediatric Cholangiocarcinoma: A Case Report.

Locally advanced cholangiocarcinoma has a poor prognosis, with long-term survival only for patients where complete surgical resection is achieved. Median overall survival with chemotherapy alone is less than 1 year. Novel strategies combining conventional chemotherapy and radiotherapy followed by targeted agents can lead to durable treatment responses and are applicable to cholangiocarcinoma management. Pediatric cholangiocarcinoma is exceedingly rare, with an estimate of 15-22 cases reported in the last 40 years. As such, no standard therapeutic regimen exists. We present a case of a 16-year-old previously healthy patient with unresectable cholangiocarcinoma whose tumor genetic sequencing revealed a novel, actionable neuregulin-1 (NRG1) gene translocation. The patient underwent standard systemic chemotherapy with gemcitabine and cisplatin followed by hypofractionated proton radiation therapy for local control. The patient then started an oral pan-ERBB (erythroblastic B receptor tyrosine kinases including ErbB1/EGFR, ErbB2/HER2, ErbB3/HER3, ErbB4/HER4) family inhibitor as a maintenance medication, remaining with stable disease and excellent quality of life for over 2 years. This case highlights a novel NRG1 fusion in a rare clinical entity that provided an opportunity to utilize a multimodal therapeutic strategy in the pediatric setting.

Comprehensive Genomic Profiling of High-Risk Pediatric Cancer Patients Has a Measurable Impact on Clinical Care.

PURPOSE: Profiling of pediatric cancers through deep sequencing of large gene panels and whole exomes is rapidly being adopted in many clinical settings. However, the most impactful approach to genomic profiling of pediatric cancers remains to be defined. METHODS: We conducted a prospective precision medicine trial, using whole-exome sequencing of tumor and germline tissue and whole-transcriptome sequencing (RNA Seq) of tumor tissue to characterize the mutational landscape of 127 tumors from 126 unique patients across the spectrum of pediatric brain tumors, hematologic malignancies, and extracranial solid tumors. RESULTS: We identified somatic tumor alterations in 121/127 (95.3%) tumor samples and identified cancer predisposition syndromes on the basis of known pathogenic or likely pathogenic germline mutations in cancer predisposition genes in 9/126 patients (7.1%). Additionally, we developed a novel scoring system for measuring the impact of tumor and germline sequencing, encompassing therapeutically relevant genomic alterations, cancer-related germline findings, recommendations for treatment, and refinement of risk stratification or prognosis. At least one impactful finding from the genomic results was identified in 108/127 (85%) samples sequenced. A recommendation to consider a targeted agent was provided for 82/126 (65.1%) patients. Twenty patients ultimately received therapy with a molecularly targeted agent, representing 24% of those who received a targeted agent recommendation and 16% of the total cohort. CONCLUSION: Paired tumor/normal whole-exome sequencing and tumor RNA Seq of de novo or relapsed/refractory tumors was feasible and clinically impactful in high-risk pediatric cancer patients.

Tumor-Type Agnostic, Targeted Therapies: BRAF Inhibitors Join the Group.

In the present review, we briefly discuss the breakthrough advances in precision medicine using a tumor-agnostic approach and focus on BRAF treatment modalities, the mechanisms of resistance and the diagnostic approach in cancers with BRAF mutations. Tumor-type agnostic drug therapies work across cancer types and present a significant novel shift in precision cancer medicine. They are the consequence of carefully designed clinical trials that showed the value of tumor biomarkers, not just in diagnosis but in therapy guidance. Six tumor-agnostic drugs (with seven indications) have been approved through October 2022 by FDA. The first tumor-agnostic treatment modality was pembrolizumab for MSI-H/dMMR solid tumors, approved in 2017. This was followed by approvals of larotrectinib and entrectinib for cancers with NTRK fusions without a known acquired resistance mutation. In 2020, pembrolizumab was approved for all TMB-high solid cancers, while a PD-L1 inhibitor dostarlimab-gxly was approved for dMMR solid cancers in 2021. A combination of BRAF/MEK inhibitors (dabrafenib/trametinib) was approved as a tumor-agnostic therapy in June 2022 for all histologic types of solid metastatic cancers harboring BRAFV600E mutations. In September 2022, RET inhibitor selpercatinib was approved for solid cancers with RET gene fusions. CONCLUSION: Precision cancer medicine has substantially improved cancer diagnostics and treatment. Tissue type-agnostic drug therapies present a novel shift in precision cancer medicine. This approach rapidly expands to provide treatments for patients with different cancers harboring the same molecular alteration.

The value of comprehensive genomic sequencing to maximize the identification of clinically actionable alterations in advanced cancer patients: a case series.

PURPOSE: We present seven cases of advanced cancer patients who initially underwent tumor testing utilizing smaller, panel-based tests, followed by a variety of therapeutic treatments which ultimately resulted in progression of their disease. These cases demonstrate the value of utilizing WES/RNA seq and characterization following disease progression in these patients and the determination of clinically targetable alterations as well as acquired resistance mutations. MATERIALS AND METHODS: All patients are part of an IRB approved observational study. WES and RNA sequencing were performed, using GEM ExTra® on tumor and blood samples obtained during routine clinical care. To accurately determine somatic versus germline alterations the test was performed with paired normal testing from peripheral blood. RESULTS: The presented cases demonstrate the clinical impact of actionable findings uncovered using GEM ExTra® in patients with advanced disease who failed many rounds of treatment. Unique alterations were identified resulting in newly identified potential targeted therapies, mechanisms of resistance, and variation in the genomic characterization of the primary versus the metastatic tumor. CONCLUSIONS: Taken together our results demonstrate that GEM ExTra® maximizes detection of actionable mutations, thus allowing for appropriate treatment selection for patients harboring both common and rare genomic alterations.

Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®.

We developed and analytically validated a comprehensive genomic profiling (CGP) assay, GEM ExTra, for patients with advanced solid tumors that uses Next Generation Sequencing (NGS) to characterize whole exomes employing a paired tumor-normal subtraction methodology. The assay detects single nucleotide variants (SNV), indels, focal copy number alterations (CNA), TERT promoter region, as well as tumor mutation burden (TMB) and microsatellite instability (MSI) status. Additionally, the assay incorporates whole transcriptome sequencing of the tumor sample that allows for the detection of gene fusions and select special transcripts, including AR-V7, EGFR vIII, EGFRvIV, and MET exon 14 skipping events. The assay has a mean target coverage of 180X for the normal (germline) and 400X for tumor DNA including enhanced probe design to facilitate the sequencing of difficult regions. Proprietary bioinformatics, paired with comprehensive clinical curation results in reporting that defines clinically actionable, FDA-approved, and clinical trial drug options for the management of the patient's cancer. GEM ExTra demonstrated analytic specificity (PPV) of > 99.9% and analytic sensitivity of 98.8%. Application of GEM ExTra to 1,435 patient samples revealed clinically actionable alterations in 83.9% of reports, including 31 (2.5%) where therapeutic recommendations were based on RNA fusion findings only.

Identifying treatment options for BRAFV600 wild-type metastatic melanoma: A SU2C/MRA genomics-enabled clinical trial.

Although combination BRAF and MEK inhibitors are highly effective for the 40-50% of cutaneous metastatic melanomas harboring BRAFV600 mutations, targeted agents have been ineffective for BRAFV600wild-type (wt) metastatic melanomas. The SU2C Genomics-Enabled Medicine for Melanoma Trial utilized a Simon two-stage optimal design to assess whether comprehensive genomic profiling improves selection of molecular-based therapies for BRAFV600wt metastatic melanoma patients who had progressed on standard-of-care therapy, which may include immunotherapy. Of the response-evaluable patients, binimetinib was selected for 20 patients randomized to the genomics-enabled arm, and nine were treated on the alternate treatment arm. Response rates for 27 patients treated with targeted recommendations included one (4%) partial response, 18 (67%) with stable disease, and eight (30%) with progressive disease. Post-trial genomic and protein pathway activation mapping identified additional drug classes that may be considered for future studies. Our results highlight the complexity and heterogeneity of metastatic melanomas, as well as how the lack of response in this trial may be associated with limitations including monotherapy drug selection and the dearth of available single and combination molecularly-driven therapies to treat BRAFV600wt metastatic melanomas.

Programmed cell death 1 (PD-1) and its ligand (PD-L1) in common cancers and their correlation with molecular cancer type.

Cancer cells expressing PD-1 ligands (PD-L1/PD-L2) inhibit immune-modulatory T-cell activation facilitating disease progression. Preliminary clinical trials exploring interruption of PD-1/PD-L1 signaling showed benefit in several cancer types. We analyzed the distribution of PD-1-positive tumor-infiltrating lymphocytes (TIL) and cancer cells' expression of PD-L1 in a molecularly profiled cohort of 437 malignancies (380 carcinomas, 33 sarcomas, and 24 melanomas). We showed that the presence of PD-1(+) TILs significantly varied among cancer types (from 0% in extraskeletal myxoid chondrosarcomas to 93% in ovarian cancer), and was generally associated with the increased number of mutations in tumor cells (P = 0.029). Cancer cell expression of PD-L1 varied from absent (in Merkel cell carcinomas) to 100% (in chondro- and liposarcomas), but showed the inverse association with the number of detected mutations (P = 0.004). Both PD-1 and PD-L1 expression were significantly higher in triple-negative breast cancers (TNBC) than in non-TNBC (P < 0.001 and 0.017, respectively). Similarly, MSI-H colon cancers had higher PD-1 and PD-L1 expression than the microsatellite stable tumors (P = 0.002 and 0.02, respectively). TP53-mutated breast cancers had significantly higher PD-1 positivity than those harboring other driver mutations (e.g., PIK3CA; P = 0.002). In non-small cell lung cancer, PD-1/PD-L1 coexpression was identified in 8 cases (19%), which lacked any other targetable alterations (e.g., EGFR, ALK, or ROS1). Our study demonstrated the utility of exploring the expression of two potentially targetable immune checkpoint proteins (PD-1/PD-L1) in a substantial proportion of solid tumors, including some aggressive subtypes that lack other targeted treatment modalities.

Comprehensive tumor profiling identifies numerous biomarkers of drug response in cancers of unknown primary site: analysis of 1806 cases.

BACKGROUND: Cancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Despite extensive laboratory and imaging efforts, the primary site usually cannot be unequivocally confirmed, and the treatment for the most part remains empirical. Recently, identification of common cancer pathway alterations in diverse cancer lineages has offered an opportunity to provide targeted therapies for patients with CUP, irrespective of the primary site. PATIENTS AND METHODS: 1806 cancers of unknown primary were identified among more than 63,000 cases profiled at Caris Life Sciences. Multiplatform profiling of the tumor samples included immunohistochemistry, gene sequencing and in situ hybridization methods in an effort to identify changes in biomarkers that are predictive of drug responses. RESULTS: Biomarkers associated with a potential drug benefit were identified in 96% of cases. Biomarkers identified included those associated with potential benefit in nearly all classes of approved cancer drugs (cytotoxic, hormonal, targeted biological drugs). Additionally, biomarkers associated with a potential lack of benefit were identified in numerous cases, which could further refine the management of patients with CUP. CONCLUSION: Comprehensive biomarker profiling of CUP may provide additional choices in treatment of patients with these difficult to treat malignancies.

RNAi screening of the kinome identifies modulators of cisplatin response in ovarian cancer cells.

OBJECTIVE: Ovarian cancer retains a poor prognosis among the female gynaecological malignancies. It constitutes about 3% of all malignancies in women and accounts for 5% of all female cancer related deaths. A standard treatment is cytoreductive surgery followed by adjuvant chemotherapy, and re-treatment with platinum based chemotherapy at the time of relapse. In order to improve cisplatin response in ovarian cancer cells, we utilized a high-throughput RNAi screening to identify kinase modulators. METHODS: A high-throughput RNAi screen was performed using a siRNA library targeting 572 kinases to identify potentiators of cisplatin response in the ovarian cancer cell line SKOV3. RESULTS: RNAi screening identified at least 55 siRNAs that potentiated the growth inhibitory effects of cisplatin in SKOV3 cells. Inhibition of ATR and CHK1 resulted in the greatest modulation of cisplatin response. Drug dose response of cisplatin in the presence of siRNA validated the effects of these target genes. To show that the siRNA data could be successfully translated into potential therapeutic strategies, CHK1 was further targeted with small molecule inhibitor PD 407824 in combination with cisplatin. Results showed that treatment of SKOV3 and OVCAR3 cells with CHK1 inhibitor PD 407824 led to sensitization of ovarian cancer cells to cisplatin. CONCLUSIONS: Our data provides kinase targets that could be exploited to design better therapeutics for ovarian cancer patients. We also demonstrate the effectiveness of high-throughput RNAi screening as a tool for identifying sensitizing targets to known and established chemotherapeutic agents.

The autophilic anti-CD20 antibody DXL625 displays enhanced potency due to lipid raft-dependent induction of apoptosis.

Despite widespread use of anti-CD20 antibodies as therapeutic agents for oncologic and autoimmune indications, precise descriptions of killing mechanisms remain incomplete. Complement-dependent cytolysis and antibody-dependent cell-mediated cytotoxicity are indicated as modes of target cell depletion; however, the importance of apoptosis induction is controversial. Studies showing that the therapeutic anti-CD20 antibody rituximab (Rituxan) mediates apoptosis of tumor cell targets in vitro after cross-linking by anti-Fc reagents suggest that enhancement strategies applied to Fc-independent activities for anti-CD20 antibodies could improve therapeutic efficacy. An anti-CD20 antibody designated DXL625, with autophilic properties such as increased binding avidity, is shown here to independently induce caspase-mediated apoptosis of an established B-cell lymphoma line in vitro. Depletion of membrane cholesterol or chelation of extracellular calcium abrogated the pro-apoptotic activity of DXL625, indicating that intact lipid rafts and calcium are required for this activity. The Fc-mediated complement-dependent and antibody-dependent cellular killing mechanisms are maintained by DXL625 despite conjugation of the parental Rituxan antibody to the autophilic DXL peptide sequence. This study shows a strategy for improving anti-CD20 immunotherapy by endowing therapeutic antibodies with self-interacting properties.

High-content siRNA screening of the kinome identifies kinases involved in Alzheimer's disease-related tau hyperphosphorylation.

BACKGROUND: Neurofibrillary tangles (NFT), a cardinal neuropathological feature of Alzheimer's disease (AD) that is highly correlated with synaptic loss and dementia severity, appear to be partly attributable to increased phosphorylation of the microtubule stabilizing protein tau at certain AD-related residues. Identifying the kinases involved in the pathologic phosphorylation of tau may provide targets at which to aim new AD-modifying treatments. RESULTS: We report results from a screen of 572 kinases in the human genome for effects on tau hyperphosphorylation using a loss of function, high-throughput RNAi approach. We confirm effects of three kinases from this screen, the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and the A-kinase anchor protein 13 (AKAP13) on tau phosphorylation at the 12E8 epitope (serine 262/serine 356). We provide evidence that EIF2AK2 effects may result from effects on tau protein expression, whereas DYRK1A and AKAP13 are likely more specifically involved in tau phosphorylation pathways. CONCLUSIONS: These findings identify novel kinases that phosphorylate tau protein and provide a valuable reference data set describing the kinases involved in phosphorylating tau at an AD-relevant epitope.

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer.

BACKGROUND: Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. METHODS: In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. RESULTS: Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. CONCLUSION: These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine.

Progression of pancreatic adenocarcinoma is significantly impeded with a combination of vaccine and COX-2 inhibition.

With a 5-year survival rate of <5%, pancreatic cancer is one of the most rapidly fatal malignancies. Current protocols for the treatment of pancreas cancer are not as effective as we desire. In this study, we show that a novel Mucin-1 (MUC1)-based vaccine in combination with a cyclooxygenase-2 inhibitor (celecoxib), and low-dose chemotherapy (gemcitabine) was effective in preventing the progression of preneoplastic intraepithelial lesions to invasive pancreatic ductal adenocarcinomas. The study was conducted in an appropriate triple transgenic model of spontaneous pancreatic cancer induced by the KRAS(G12D) mutation and that expresses human MUC1 as a self molecule. The combination treatment elicited robust antitumor cellular and humoral immune responses and was associated with increased apoptosis in the tumor. The mechanism for the increased immune response was attributed to the down-regulation of circulating prostaglandin E(2) and indoleamine 2, 3,-dioxygenase enzymatic activity, as well as decreased levels of T regulatory and myeloid suppressor cells within the tumor microenvironment. The preclinical data provide the rationale to design clinical trials with a combination of MUC1-based vaccine, celecoxib, and gemcitabine for the treatment of pancreatic cancer.

MUC1 enhances tumor progression and contributes toward immunosuppression in a mouse model of spontaneous pancreatic adenocarcinoma.

MUC1, a membrane tethered mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic adenocarcinoma. However, the role of MUC1 in pancreatic cancer has been elusive, partly due to the lack of an appropriate model. We report the characterization of a novel mouse model that expresses human MUC1 as a self molecule (PDA.MUC1 mice). Pancreatic tumors arise in an appropriate MUC1-tolerant background within an immune-competent host. Significant enhancement in the development of pancreatic intraepithelial preneoplastic lesions and progression to adenocarcinoma is observed in PDA.MUC1 mice, possibly due to increased proliferation. Tumors from PDA.MUC1 mice express higher levels of cyclooxygenase-2 and IDO compared with PDA mice lacking MUC1, especially during early stages of tumor development. The increased proinflammatory milieu correlates with an increased percentage of regulatory T cells and myeloid suppressor cells in the pancreatic tumor and tumor draining lymph nodes. Data shows that during pancreatic cancer progression, MUC1-mediated mechanisms enhance the onset and progression of the disease, which in turn regulate the immune responses. Thus, the mouse model is ideally suited for testing novel chemopreventive and therapeutic strategies against pancreatic cancer.

Functional evidence implicating S100P in prostate cancer progression.

S100P protein regulates calcium signal transduction and mediates cytoskeletal interaction, protein phosphorylation and transcriptional control. We have previously shown how elevated S100P levels in prostate cancer strongly correlate with progression to metastatic disease. In our study, we evaluated the functional significance of S100P expression on prostate tumor growth in vitro and in vivo. S100P levels were modulated by overexpressing S100P in PC3 prostate cancer cells and by silencing S100P levels in 22Rv1 prostate cancer cells. Overexpression of S100P in PC3 cells promoted cell growth, increased the percentage of S-phase cells, decreased basal apoptosis rate and promoted anchorage independent growth in soft agar. Furthermore, prostate cancer cells overexpressing S100P were protected against camptothecin-induced apoptosis. Conversely, silencing of S100P in 22Rv1 cells using siRNA resulted in a prominent cytostatic effect. The influence of S100P on tumor growth and metastases were assessed in vivo. S100P-overexpressing PC3 cells had a dramatically increased tumor formation compared to controls. Microarray analysis showed the involvement of growth pathways including increased androgen receptor expression in S100P-overexpressing cells. These results provide the first functional proof that S100P overexpression can upregulate androgen receptor expression and thereby promote prostate cancer progression by increasing cell growth. Moreover, the results confirm the oncogenic nature of S100P in prostate cancer and suggest that the protein may directly confer resistance to chemotherapy. Hence, S100P could be considered a potential drug target or a chemosensitization target, and could also serve as a biomarker for aggressive, hormone-refractory and metastatic prostate cancer.

Intersex-like (IXL) is a cell survival regulator in pancreatic cancer with 19q13 amplification.

Pancreatic cancer is a highly aggressive disease characterized by poor prognosis and vast genetic instability. Recent microarray-based, genome-wide surveys have identified multiple recurrent copy number aberrations in pancreatic cancer; however, the target genes are, for the most part, unknown. Here, we characterized the 19q13 amplicon in pancreatic cancer to identify putative new drug targets. Copy number increases at 19q13 were quantitated in 16 pancreatic cancer cell lines and 31 primary tumors by fluorescence in situ hybridization. Cell line copy number data delineated a 1.1 Mb amplicon, the presence of which was also validated in 10% of primary pancreatic tumors. Comprehensive expression analysis by quantitative real-time reverse transcription-PCR indicated that seven transcripts within this region had consistently elevated expression levels in the amplified versus nonamplified cell lines. High-throughput loss-of-function screen by RNA interference was applied across the amplicon to identify genes whose down-regulation affected cell viability. This screen revealed five genes whose down-regulation led to significantly decreased cell viability in the amplified PANC-1 cells but not in the nonamplified MiaPaca-2 cells, suggesting the presence of multiple biologically interesting genes in this region. Of these, the transcriptional regulator intersex-like (IXL) was consistently overexpressed in amplified cells and had the most dramatic effect on cell viability. IXL silencing also resulted in G(0)-G(1) cell cycle arrest and increased apoptosis in PANC-1 cells. These findings implicate IXL as a novel amplification target gene in pancreatic cancer and suggest that IXL is required for cancer cell survival in 19q13-amplified tumors.

A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells.

INTRODUCTION: Cyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor. METHODS: To determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2. RESULTS: We found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E2. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation. CONCLUSION: Antivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.

Cyclooxygenase-2 inhibitor enhances the efficacy of a breast cancer vaccine: role of IDO.

We report that administration of celecoxib, a specific cyclooxygenase-2 (COX-2) inhibitor, in combination with a dendritic cell-based cancer vaccine significantly augments vaccine efficacy in reducing primary tumor burden, preventing metastasis, and increasing survival. This combination treatment was tested in MMTV-PyV MT mice that develop spontaneous mammary gland tumors with metastasis to the lungs and bone marrow. Improved vaccine potency was associated with an increase in tumor-specific CTLs. Enhanced CTL activity was attributed to a significant decrease in levels of tumor-associated IDO, a negative regulator of T cell activity. We present data suggesting that inhibiting COX-2 activity in vivo regulates IDO expression within the tumor microenvironment; this is further corroborated in the MDA-MB-231 human breast cancer cell line. Thus, a novel mechanism of COX-2-induced immunosuppression via regulation of IDO has emerged that may have implications in designing future cancer vaccines.

Mechanisms underlying the growth inhibitory effects of the cyclo-oxygenase-2 inhibitor celecoxib in human breast cancer cells.

INTRODUCTION: Inhibitors of cyclo-oxygenase (COX)-2 are being extensively studied as anticancer agents. In the present study we evaluated the mechanisms by which a highly selective COX-2 inhibitor, celecoxib, affects tumor growth of two differentially invasive human breast cancer cell lines. METHODS: MDA-MB-231 (highly invasive) and MDA-MB-468 (moderately invasive) cell lines were treated with varying concentrations of celecoxib in vitro, and the effects of this agent on cell growth and angiogenesis were monitored by evaluating cell proliferation, apoptosis, cell cycle arrest, and vasculogenic mimicry. The in vitro results of MDA-MB-231 cell line were further confirmed in vivo in a mouse xenograft model. RESULTS: The highly invasive MDA-MB-231 cells express higher levels of COX-2 than do the less invasive MDA-MB-468 cells. Celecoxib treatment inhibited COX-2 activity, indicated by prostaglandin E2 secretion, and caused significant growth arrest in both breast cancer cell lines. In the highly invasive MDA-MB-231 cells, the mechanism of celecoxib-induced growth arrest was by induction of apoptosis, associated with reduced activation of protein kinase B/Akt, and subsequent activation of caspases 3 and 7. In the less invasive MDA-MB-468 cells, growth arrest was a consequence of cell cycle arrest at the G0/G1 checkpoint. Celecoxib-induced growth inhibition was reversed by addition of exogenous prostaglandin E2 in MDA-MB-468 cells but not in MDA-MB-231 cells. Furthermore, MDA-MB-468 cells formed significantly fewer extracellular matrix associated microvascular channels in vitro than did the high COX-2 expressing MDA-MB-231 cells. Celecoxib treatment not only inhibited cell growth and vascular channel formation but also reduced vascular endothelial growth factor levels. The in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of celecoxib significantly reduced tumor growth of MDA-MB-231 cells, which was associated with reduced vascularization and increased necrosis in the tumor mass. CONCLUSION: The disparate molecular mechanisms of celecoxib-induced growth inhibition in human breast cancer cells depends upon the level of COX-2 expression and the invasive potential of the cell lines examined. Data suggest a role for COX-2 not only in the growth of cancer cells but also in activating the angiogenic pathway through regulating levels of vascular endothelial growth factor.