Mourad Tighiouart

Insulin-like Growth Factor-I–Dependent Up-regulation of ZEB1 Drives Epithelial-to-Mesenchymal Transition in Human Prostate Cancer Cells

The epithelial-to-mesenchymal transition (EMT) is crucial for the migration and invasion of many epithelial tumors, including prostate cancer. Although it is known that ZEB1 overexpression promotes EMT primarily through down-regulation of E-cadherin in a variety of cancers, the soluble ligands responsible for the activation of ZEB1 have yet to be identified. In the present study, we investigated the role of insulin-like growth factor-I (IGF-I) in the regulation of ZEB1 during EMT associated with prostate tumor cell migration. We found that ZEB1 is expressed in highly aggressive prostate cancer cells and that its expression correlates directly with Gleason grade in human prostate tumors (P < 0.001). IGF-I up-regulates ZEB1 expression in prostate cancer cells exhibiting an epithelial phenotype. In prostate cancer cells displaying a mesenchymal phenotype, ZEB1 inhibition reverses the suppression of E-cadherin protein and down-regulates the expression of the mesenchymal markers N-cadherin and fibronectin. Furthermore, ZEB1 blockade decreases migratory and invasive potential in ARCaP(M) compared with the control. These results identify ZEB1 as a key transcriptional regulator of EMT in prostate cancer and suggest that the aberrant expression of ZEB1 in prostate cancer cells occurs in part in response to IGF-I stimulation.

BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization.

BRCA1-associated protein-1 (BAP1), a deubiquitinating enzyme of unknown cellular function, is mutated in breast and lung cancers. In this study, we have shown for the first time that BAP1 has tumor suppressor activity in vivo by showing that BAP1 can suppress tumorigenicity of lung cancer cells in athymic nude mice. We show that BAP1 fulfills another criterion of a genuine tumor suppressor because cancer-associated BAP1 mutants are deficient in deubiquitinating activity. We show for the first time that one of the two predicted nuclear targeting motifs is required for nuclear localization of BAP1 and that a truncation mutant found in a lung cancer cell line results in BAP1 that fails to localize to the nucleus. Furthermore, we show that deubiquitinating activity and nuclear localization are both required for BAP1-mediated tumor suppression in nude mice. We show that BAP1 exerts its tumor suppressor functions by affecting the cell cycle, speeding the progression through the G(1)-S checkpoint, and inducing cell death via a process that has characteristics of both apoptosis and necrosis. Surprisingly, BAP1-mediated growth suppression is independent of wild-type BRCA1. Because deubiquitinating enzymes are components of the ubiquitin proteasome system, this pathway has emerged as an important target for anticancer drugs. The identification of the deubiquitinating enzyme BAP1 as a tumor suppressor may lead to further understanding of how the ubiquitin proteasome system contributes to cancer and aid in the identification of new targets for cancer therapy.

Translation of Innovative Designs Into Phase I Trials

PURPOSE Phase I clinical trials of new anticancer therapies determine suitable doses for further testing. Optimization of their design is vital in that they enroll cancer patients whose well-being is distinctly at risk. This study examines the effectiveness of knowledge transfer about more effective statistical designs to clinical practice. METHODS We examined abstract records of cancer phase I trials from the Science Citation Index database between 1991 and 2006 and classified them into clinical (dose-finding trials) and statistical trials (methodologic studies of dose-escalation designs). We then mapped these two sets by tracking which trials adopted new statistical designs. RESULTS One thousand two hundred thirty-five clinical and 90 statistical studies were identified. Only 1.6% of the phase I cancer trials (20 of 1,235 trials) followed a design proposed in one of the statistical studies. These 20 clinical studies showed extensive lags between publication of the statistical paper and its translation into a clinical paper. These 20 clinical trials followed Bayesian adaptive designs. The remainder used variations of the standard up-and-down method. CONCLUSION A consequence of using less effective designs is that more patients are treated with doses outside the therapeutic window. Simulation studies have shown that up-and-down designs treated only 35% of patients at optimal dose levels versus 55% for Bayesian adaptive designs. This implies needless loss of treatment efficacy and, possibly, lives. We suggest that regulatory agencies (eg, US Food and Drug Administration) should proactively encourage the adoption of statistical designs that would allow more patients to be treated at near-optimal doses while controlling for excessive toxicity.

HFT-T, a Targeting Nanoparticle, Enhances Specific Delivery of Paclitaxel to Folate Receptor-Positive Tumors

Nonspecific distribution of chemotherapeutic drugs (such as paclitaxel) is a major factor contributing to side effects and poor clinical outcomes in the treatment of human head and neck cancer. To develop novel drug delivery systems with enhanced efficacy and minimized adverse effects, we synthesized a ternary conjugate heparin-folic acid-paclitaxel (HFT), loaded with additional paclitaxel (T). The resulting nanoparticle, HFT-T, is expected to retain the antitumor activity of paclitaxel and specifically target folate receptor (FR)-expressing tumors, thereby increasing the bioavailability and efficacy of paclitaxel. In vitro experiments found that HFT-T selectively recognizes FR-positive human head and neck cancer cell line KB-3-1, displaying higher cytotoxicity compared to the free form of paclitaxel. In a subcutaneous KB-3-1 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably improved antitumor efficacy of paclitaxel. The average tumor volume in the HFT-T treatment group was 92.9 +/- 78.2 mm(3) vs 1670.3 +/- 286.1 mm(3) in the mice treated with free paclitaxel. Furthermore, paclitaxel tumors showed a resurgence of growth after several weeks of treatment, but this was not observed with HFT-T. This indicates that HFT-T could be more effective in preventing tumors from developing drug resistance. No significant acute in vivo toxicity was observed. These results indicate that specific delivery of paclitaxel with a ternary structured nanoparticle (HFT-T) targeting FR-positive tumor is a promising strategy to enhance chemotherapy efficacy and minimize adverse effects.

Withaferin A inhibits breast cancer invasion and metastasis at sub-cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation

Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition and reduced patient survival. As vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data showed that WFA had weak cytotoxic and apoptotic activity at concentrations less than or equal to 500 nM, but retained potent anti‐invasive activity at these low doses. Imaging of breast cancer cell lines revealed that WFA induces perinuclear vimentin accumulation followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin‐binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti‐invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2 μM in plasma with a half‐life of 1.36 hr following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose‐dependent inhibition of metastatic lung nodules and induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti‐metastatic agent and the anti‐metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.

A Collagen-Remodeling Gene Signature Regulated by TGF-β Signaling Is Associated with Metastasis and Poor Survival in Serous Ovarian Cancer

Purpose: To elucidate molecular pathways contributing to metastatic cancer progression and poor clinical outcome in serous ovarian cancer. Experimental Design: Poor survival signatures from three different serous ovarian cancer datasets were compared and a common set of genes was identified. The predictive value of this gene signature was validated in independent datasets. The expression of the signature genes was evaluated in primary, metastatic, and/or recurrent cancers using quantitative PCR and in situ hybridization. Alterations in gene expression by TGF-β1 and functional consequences of loss of COL11A1 were evaluated using pharmacologic and knockdown approaches, respectively. Results: We identified and validated a 10-gene signature (AEBP1, COL11A1, COL5A1, COL6A2, LOX, POSTN, SNAI2, THBS2, TIMP3, and VCAN) that is associated with poor overall survival (OS) in patients with high-grade serous ovarian cancer. The signature genes encode extracellular matrix proteins involved in collagen remodeling. Expression of the signature genes is regulated by TGF-β1 signaling and is enriched in metastases in comparison with primary ovarian tumors. We demonstrate that levels of COL11A1, one of the signature genes, continuously increase during ovarian cancer disease progression, with the highest expression in recurrent metastases. Knockdown of COL11A1 decreases in vitro cell migration, invasion, and tumor progression in mice. Conclusion: Our findings suggest that collagen-remodeling genes regulated by TGF-β1 signaling promote metastasis and contribute to poor OS in patients with serous ovarian cancer. Our 10-gene signature has both predictive value and biologic relevance and thus may be useful as a therapeutic target. Clin Cancer Res; 20(3); 711–23. ©2013 AACR.

A folate receptor-targeting nanoparticle minimizes drug resistance in a human cancer model.

Resistance to chemotherapy is a major obstacle in cancer therapy. The main purpose of this study is to evaluate the potential of a folate receptor-targeting nanoparticle to overcome/minimize drug resistance and to explore the underlying mechanisms. This is accomplished with enhanced cellular accumulation and retention of paclitaxel (one of the most effective anticancer drugs in use today and a well-known P-glycoprotein (P-gp) substrate) in a P-gp-overexpressing cancer model. The folate receptor-targeted nanoparticle, HFT-T, consists of a heparin-folate-paclitaxel (HFT) backbone with an additional paclitaxel (T) loaded in its hydrophobic core. In vitro analyses demonstrated that the HFT-T nanoparticle was superior to free paclitaxel or nontargeted nanoparticle (HT-T) in inhibiting proliferation of P-gp-overexpressing cancer cells (KB-8-5), partially due to its enhanced uptake and prolonged intracellular retention. In a subcutaneous KB-8-5 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably prolonged retention within tumor tissues. Importantly, HFT-T treatment markedly retarded tumor growth in a xenograft model of resistant human squamous cancer. Immunohistochemical analysis further indicated that increased in vivo efficacy of HFT-T nanoparticles was associated with a higher degree of microtubule stabilization, mitotic arrest, antiangiogenic activity, and inhibition of cell proliferation. These findings suggest that when the paclitaxel was delivered as an HFT-T nanoparticle, the drug is better retained within the P-gp-overexpressing cells than the free form of paclitaxel. These results indicated that the targeted HFT-T nanoparticle may be promising in minimizing P-gp related drug resistance and enhancing therapeutic efficacy compared with the free form of paclitaxel.

A Phase I/II Trial Combining High-Dose Melphalan and Autologous Transplant with Bortezomib for Multiple Myeloma: A Dose- and Schedule-Finding Study

Purpose: We did a randomized phase I/II trial designed to evaluate the safety and efficacy of combining the proteasome inhibitor bortezomib with high-dose melphalan as the conditioning for high-dose therapy and autologous transplant for myeloma. Experimental Design: Enrolled patients were limited to those who did not achieve a very good partial remission (VGPR) following one or more induction regimens, and were randomized to receive a single escalating dose of bortezomib (1.0, 1.3, or 1.6 mg/m2) either 24 hours before or 24 hours after high-dose melphalan. Dose escalation was based on the escalation with overdose control (EWOC), a Bayesian statistical model. Bone marrow aspirates were collected before initiation of therapy and at the time of transplant to evaluate which sequence resulted in maximal plasma cell apoptosis, and response to transplant was assessed by the International Myeloma Working Group criteria. Results: Among 39 randomized patients, 20 received bortezomib after melphalan and 19 received bortezomib before melphalan. Toxicities and posttransplant hematopoietic recovery rates were similar between arms. The overall response rate for all patients was 87%, with 51% achieving a VGPR or better. Pharmacodynamic studies showed greater plasma cell apoptosis among patients who received bortezomib following melphalan. Conclusions: The use of bortezomib in conjunction with high-dose melphalan is safe, with data suggesting improved efficacy. A single dose of bortezomib administered after high-dose melphalan is the recommended dose and schedule for future clinical investigation. Clin Cancer Res; 16(20); 5079–86. ©2010 AACR.

Primary mucosal melanoma of the head and neck. Comparison of clinical presentation and histopathologic features of oral and sinonasal melanoma

We reviewed all cases of head and neck mucosal melanomas (HNMM) treated at Emory University and affiliated hospitals during a 20 year period and evaluate overall survival, recurrences and efficacy of treatment. Comparisons were made between sinonasal melanoma (SNM) and oral cavity melanoma (OCM) including clinical features, histopathologic features, treatment, and clinical outcomes. We analyzed pathologic features and clinical outcomes of 22 cases of primary SNM and eight cases of OCM treated at Emory University Hospital between 1986 and 2006. Sixteen patients were stage I (53%), nine patients were stage II (30%) and five patients were stage III (17%). Mean age was 67.5 years with a range from 32 to 85 years. Sixty percent were men and 92% were white. The average follow-up time was 22.1 months and the median was 15.5 months. The average delay between onset of symptoms and diagnosis was 5.6 months. Patients with SNM had a lower incidence of nodal metastasis at initial presentation when compared with OCM (22% vs. 50%) (p<.08). Surgical resection was the primary treatment and was performed in 27/30 patients (90%). Two patients with SNM and 1 patient with OCM, because of advanced stage at diagnosis had no treatment. Radiation was used in 50% (15) of the patients and radiation as postoperative therapy was administered to 44.8% (13) of the patients. The combination of surgery, radiation and adjuvant therapy was administered to 39.2% (11/2 8) of the patients. The recurrence rate at the time of closing this study was 43.3% (13 patients) with a mean time for development of recurrences of 13.1 months. Twenty-five patients (83%) died during the course of this study. Nineteen deaths (82%) corresponded to patients with SNM and six deaths (75%) to patients with OCM. The overall survival mean time was 21 months (median 12.5) with a range between 1 and 143 months. The 1, 2, 3, and 5 year survival rate for this study was 53.3%, 40%, 15% and 10%, respectively. The oral cavity was the location for the two patients still alive after 5 years. Although survival time correlated with Stage, particularly Stage 1, this was not statistically significant. Survival time did not correlate with surgery or adjuvant therapy. Statistically significant differences were noted between the pathologic features of OCM and SNM. These significant pathologic differences did not correlate with disease specific survival: OCM and SNM (median survival, 17 months vs. 12 months). Mucosal melanoma of the head and neck is a rare entity. Unfortunately, most patients present with advanced local disease. Local, regional recurrences and distant metastasis still occur despite the implementation of aggressive treatment, including surgery, radiation and adjuvant therapy. Despite significant pathologic differences between SNM and OCM, no survival advantage was seen.

Synergistic inhibition of head and neck tumor growth by green tea (−)-epigallocatechin-3-gallate and EGFR tyrosine kinase inhibitor

One of the mechanisms of the antitumor activity of green tea (−)‐epigallocatechin‐3‐gallate (EGCG) is associated with its effect on epidermal growth factor receptor (EGFR)‐mediated signaling transduction pathways. We investigated whether combining EGCG with the EGFR‐tyrosine kinase inhibitor (EGFR‐TKI) erlotinib may augment erlotinib‐induced cell growth inhibition of squamous cell carcinoma of the head and neck (SCCHN) in a mouse xenograft model. In vitro studies with 5 head and neck cancer cell lines revealed that synergistic cell growth inhibition by the combination of EGCG and erlotinib was associated with significantly greater inhibition of pEGFR and pAKT, increased activation of caspases 9, 3 and PARP compared to the inhibition induced by EGCG or erlotinib alone. Erlotinib inhibited phosphorylation of EGFR, stabilizing EGFR at the plasma membrane, whereas EGCG induced EGFR internalization and ubiquitin‐degradation, ultimately undermining EGFR signaling. The efficacy of the combination treatment was investigated with nude mice (n = 25) orally gavaged with vehicle control, EGCG, erlotinib or the combination at the same doses for 7 days, followed by subcutaneous injection with Tu212 cells. Animals were continuously administered the agents 5 days weekly for 7 weeks. The combined treatment resulted in significantly greater inhibition of tumor growth and delayed tumor progression as a result of increased apoptosis, decreased cell proliferation and reduced pEGFR and pAKT compared to the single agent treatment groups. Our results suggest a synergistic antitumor effect of a combined treatment with EGCG and erlotinib, and provide a promising regimen for future chemoprevention and treatment of SCCHN.