Rajasubramaniam Shanmugam

Nuclear Factor-κB Is Constitutively Activated in Prostate Cancer In vitro and Is Overexpressed in Prostatic Intraepithelial Neoplasia and Adenocarcinoma of the Prostate

Purpose: The transcription factor nuclear factor-κB (NF-κB) promotes the production of angiogenic, antiapoptotic, and prometastatic factors that are involved in carcinogenesis. Experimental Design: Electromobility gel shift assays were used to evaluate NF-κB DNA binding in vitro. The functional relevance of NF-κB DNA binding was assessed by both cDNA array analyses and proliferation assays of prostate cancer cells with and without exposure to an NF-κB inhibitor, parthenolide. Immunohistochemistry staining for the p65 NF-κB subunit was used to determine the frequency and location of NF-κB in 97 prostatectomy specimens. The amount of staining was quantified on a 0–3+ scale. Results: An electromobility gel shift assay confirmed the presence of NFκB DNA binding in all four prostate cancer cell lines tested. The binding was inhibited by parthenolide, and this agent also decreased multiple gene transcripts under the control of NF-κB and inhibited proliferation of prostate cancer cells. The staining results revealed overexpression of p65 in the prostatic intraepithelial neoplasia and cancer compared with the benign epithelium. Specifically, there was a predominance of 1+ and 2+ with no 3+ staining in benign epithelium, whereas there was only 2+ and 3+ staining (30 and 70%, respectively) in the cancerous areas. These differences were statistically different. There was no correlation with tumor grade or stage. Conclusions: NF-κB is constitutively activated in prostate cancer and functionally relevant in vitro. Immunohistochemistry of human prostatectomy specimens demonstrated overexpression of the active subunit of NF-κB, p65, and that this occurs at an early stage in the genesis of prostate cancer. This work supports the rationale for targeting NF-κB for the prevention and/or treatment of prostate cancer.

Antimyeloma Effects of a Sesquiterpene Lactone Parthenolide

Purpose: Nuclear factor-κB (NF-κB), activated in multiple myeloma (MM) cells by microenvironmental cues, confers resistance to apoptosis. The sesquiterpene lactone parthenolide targets NF-κB. However, its therapeutic potential in MM is not known. Experimental Designs: We explored the effects of parthenolide on MM cells in the context of the bone marrow microenvironment. Results: Parthenolide inhibited growth of MM cells lines, including drug-resistant cell lines, and primary cells in a dose-dependent manner. Parthenolide overcame the proliferative effects of cytokines interleukin-6 and insulin-like growth factor I, whereas the adhesion of MM cells to bone marrow stromal cells partially protected MM cells against parthenolide effect. In addition, parthenolide blocked interleukin-6 secretion from bone marrow stromal cells triggered by the adhesion of MM cells. Parthenolide cytotoxicity is both caspase-dependent and caspase-independent. Parthenolide rapidly induced caspase activation and cleavage of PARP, MCL-1, X-linked inhibitor of apoptosis protein, and BID. Parthenolide rapidly down-regulated cellular FADD-like IL-1β–converting enzyme inhibitory protein, and direct targeting of cellular FADD-like IL-1β–converting enzyme inhibitory protein using small interfering RNA oligonucleotides inhibited MM cell growth and lowered the parthenolide concentration required for growth inhibition. An additive effect and synergy were observed when parthenolide was combined with dexamethasone and TNF-related apoptosis-inducing ligand, respectively. Conclusion: Collectively, parthenolide has multifaceted antitumor effects toward both MM cells and the bone marrow microenvironment. Our data support the clinical development of parthenolide in MM therapy.

A Noncanonical Flt3ITD/NF-κB Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia

Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB–and c-Jun–responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD+ cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD–TAK1–DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results: AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD+ AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD+ human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB–inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ AMLs had selective nuclear activation of p52NF-κB. Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ AML. Clin Cancer Res; 18(2); 360–9. ©2011 AACR.

A phase II study of pemetrexed as second-line chemotherapy for the treatment of metastatic castrate-resistant prostate cancer (CRPC); Hoosier Oncology Group GU03-67

BACKGROUND No standard therapy exists for post-docetaxel castrate-resistant prostate cancer (CRPC) patients. This trial aimed to determine the safety and efficacy of pemetrexed in post-docetaxel CRPC patients. MATERIALS AND METHODS CRPC patients with progression after docetaxel (Taxotere) therapy received pemetrexed (500 mg/m2) i.v. every 3 weeks. The primary end point was prostate-specific antigen (PSA) response. A pharmacogenetic analysis of the reduced folate carrier-1 gene (RFC1) G80A polymorphism was also carried out. RESULTS Forty-nine patients were enrolled: median age 68 years, median baseline PSA 72 ng/ml, and median Karnofsky performance status of 90. Grade 3 or 4 toxicity occurred in 20 (43%) and four patients (8%), respectively. Confirmed >50% PSA decline occurred in four patients (8%), stable PSA lasting at least 12 weeks in 10 patients (20%). A significant relationship was observed between time from prior docetaxel therapy and overall survival. Pharmacogenetic analyses of RFC1 G80A genotype frequencies showed no relationship between genotypes and clinical efficacy. CONCLUSIONS Pemetrexed treatment of CRPC patients after docetaxel therapy was associated with only modest clinical activity. Further investigation of pemetrexed as a single agent in a nonenriched CRPC population is unlikely to add significant clinical benefit over that seen with traditional second-line chemotherapy agents such as mitoxantrone.