Alan So

Heat Shock Protein 27 Increases after Androgen Ablation and Plays a Cytoprotective Role in Hormone-Refractory Prostate Cancer

Heat shock protein 27 (Hsp27) is a chaperone implicated as an independent predictor of clinical outcome in prostate cancer. Our aim was to characterize changes in Hsp27 after androgen withdrawal and during androgen-independent progression in prostate xenografts and human prostate cancer to assess the functional significance of these changes using antisense inhibition of Hsp27. A tissue microarray was used to measure changes in Hsp27 protein expression in 232 specimens from hormone naive and posthormone-treated cancers. Hsp27 expression was low or absent in untreated human prostate cancers but increased beginning 4 weeks after androgen-ablation to become uniformly highly expressed in androgen-independent tumors. Androgen-independent human prostate cancer PC-3 cells express higher levels of Hsp27 mRNA in vitro and in vivo, compared with androgen-sensitive LNCaP cells. Phosphorothioate Hsp27 antisense oligonucleotides (ASOs) and small interference RNA potently inhibit Hsp27 expression, with increased caspase-3 cleavage and PC3 cell apoptosis and 87% decreased PC3 cell growth. Hsp27 ASO and small interference RNA also enhanced paclitaxel chemosensitivity in vitro, whereas in vivo, systemic administration of Hsp27 ASO in athymic mice decreased PC-3 tumor progression and also significantly enhanced paclitaxel chemosensitivity. These findings suggest that increased levels of Hsp27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression.

Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced Growth Ability, and Cell Sensitization to Genotoxic and Oxidative Stress

Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU’s biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H2O2, respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.

Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells

Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer. [Mol Cancer Ther 2007;6(1):299–308]

Small interference RNA targeting heat-shock protein 27 inhibits the growth of prostatic cell lines and induces apoptosis via caspase-3 activation in vitro

To evaluate synthetic small interference RNA (siRNA) compounds targeting heat‐shock protein 27 (Hsp27) as an alternative approach to Hsp27 ‘knockdown’ in prostate cancer cells, as Hsp27 expression is highly up‐regulated in prostate cancer cells after androgen withdrawal or chemotherapy, to become uniformly highly expressed in androgen‐independent (AI) prostate cancer.

Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27–eIF4E interaction may serve as a therapeutic target in advanced PC.

Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells both in vitro and in vivo

Clusterin is a stress-associated cytoprotective chaperone up-regulated by various apoptotic triggers in many cancers and confers treatment resistance when overexpressed. The objectives of this study were to evaluate clusterin expression levels in human breast cancer and to determine whether antisense oligonucleotides or double-stranded small interfering RNAs (siRNA) targeting the clusterin gene enhance apoptosis induced by paclitaxel. Clusterin immunostaining was evaluated in a tissue microarray of 379 spotted breast cancers. The effect of hormone withdrawal, paclitaxel treatment, clusterin antisense oligonucleotide (OGX-011), and siRNA treatments on clusterin expression was examined in MCF-7 and MDA-MB-231 cells. Northern, quantitative real-time PCR, and Western analyses were used to measure change in clusterin mRNA and protein levels. The effect of OGX-011 or siRNA clusterin treatment on chemosensitivity to paclitaxel was done in both cell lines in vitro, whereas the ability of OGX-011 to chemosensitize in vivo was evaluated in athymic mice bearing MCF-7 tumors. Clusterin was expressed in 62.5% of tumors within the tissue microarray. Clusterin expression increased after estrogen withdrawal and paclitaxel treatment in vitro in MCF-7 cells. OGX-011 or siRNA clusterin decreased clusterin levels by >90% in a dose-dependent, sequence-specific manner and significantly enhanced chemosensitivity to paclitaxel in vitro. When combined, OGX-011 or siRNA clusterin reduced the IC50 by 2-log compared with paclitaxel alone. In vivo administration of OGX-011 enhanced the effects of paclitaxel to significantly delay MCF-7 tumor growth. These data identify clusterin as a valid therapeutic target and provides preclinical proof-of-principle to test OGX-011 in multimodality therapies for breast cancer. [Mol Cancer Ther 2005;4(12):1837–49]

Active Surveillance Is the Preferred Approach to Clinical Stage I Testicular Cancer

Craig R. Nichols, Virginia Mason Medical Center, Seattle, WA Bruce Roth, Washington University School of Medicine, St Louis, MO Peter Albers, University Hospital Heinrich-Heine, University of Dusseldorf, Dusseldorf, Germany Lawrence H. Einhorn and Richard Foster, Melvin and Bren Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN Siamak Daneshmand, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA Michael Jewett and Padraig Warde, Princess Margaret Hospital, University of Toronto, Toronto, Ontario, Canada Christopher J. Sweeney and Clair Beard, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Boston, MA Tom Powles, Bart’s Cancer Institute, St Bartholomew’s Hospital, Queen Mary University of London, London, United Kingdom Scott Tyldesley and Alan So, British Columbia Cancer Agency–Vancouver Cancer Centre, University of British Columbia, Vancouver, British Columbia, Canada Christopher Porter and Semra Olgac, Virginia Mason Medical Center, Seattle, WA Karim Fizazi, Institute Gustave Roussy, University of Paris Sud, Paris, France Brandon Hayes-Lattin, Knight Cancer Institute, Oregon Health and Science University, Portland, OR Peter Grimison, Royal Prince Alfred Hospital, Sydney Cancer Centre, University of Sydney, Sydney, New South Wales, Australia Guy Toner, Peter MacCallum Cancer Center, University of Melbourne, Melbourne, Victoria, Australia Richard Cathomas, Kantonsspital Graubuenden, Chur, Switzerland Carsten Bokemeyer, University Medical Centre Eppendorf, Hamburg University, Hamburg, Germany Christian Kollmannsberger, British Columbia Cancer Agency–Vancouver Cancer Centre, University of British Columbia, Vancouver, British Columbia, Canada

Management of Disseminated Nonseminomatous Germ Cell Tumors With Risk-Based Chemotherapy Followed by Response-Guided Postchemotherapy Surgery

PURPOSE The management of patients with a radiographic complete response after chemotherapy remains controversial. The current study assesses the outcome for a modern, unselected patient population with disseminated testicular cancer with particular emphasis on those achieving a radiographic complete remission to combination chemotherapy. PATIENTS AND METHODS All patients with disseminated nonseminoma seen between 1999 and 2007 at the British Columbia Cancer Agency (BCCA) as well as through the Oregon Testis Cancer Program were retrospectively reviewed. A total of 276 patients treated with combination chemotherapy were identified. A radiographic complete remission (CR) was defined as disappearance of all metastatic lesions or minimal residual tissue <or= 1 cm. RESULTS One hundred sixty-one patients achieved a CR. Results for the total population and CR subset were as follows: International Germ Cell Cancer Consensus Group stage good/intermediate/poor 84%/5%/11% (CR subset, 94%/3%/3%), presence of teratoma in the primary tumor 40% (CR subset, 55%), relapses 13%, death from disease 3% (CR subset, 6% and 0%, respectively). Two of the total 10 relapses in the CR group occurred beyond 2 years. Eight of the 10 relapses in the CR group were treated surgically for teratoma alone, whereas two required salvage chemotherapy. Disease-specific survival for the CR group was 100% after a median follow-up of 52 months (range, 3 to 135 months). CONCLUSION Modern risk-adapted systemic chemotherapy with or without surgery for current populations of patients with disseminated testicular nonseminoma results in superb outcomes. Patients with disseminated germ cell tumors who obtain a complete serologic remission and no or minimal radiographic residual can be safely observed without adjunctive regional surgery.

ASAP1, a Gene at 8q24, Is Associated with Prostate Cancer Metastasis

Metastatic prostate cancer is a terminal disease, and the development of reliable prognostic tools and more effective therapy is critically important for improved disease survival and management. This study was aimed at identifying genes that are differentially expressed in metastatic and nonmetastatic prostate cancer cells and, as such, could be critical in the development of metastasis. Long-SAGE analysis was used to compare a transplantable human metastatic prostate cancer subline, PCa1-met, with a nonmetastatic counterpart, PCa2. Both sublines were developed from a patients prostate cancer specimen via subrenal capsule grafting and subsequent orthotopic implantation into SCID mice. Among various differentially expressed genes identified, ASAP1, an 8q24 gene encoding an ADP-ribosylation factor GTPase-activating protein not previously associated with prostate cancer, was up-regulated in the metastatic subline as confirmed by quantitative real-time PCR. Immunohistochemistry of xenograft sections showed that cytoplasmic ASAP1 protein staining was absent or weak in benign tissue, significantly stronger in nonmetastatic PCa2 tissue, and strongest in PCa1-met tissue. In clinical specimens, ASAP1 protein staining was elevated in 80% of primary prostate cancers and substantially higher in metastatic lesions compared with benign prostate tissue. Moreover, additional ASAP1 gene copies were detected in 58% of the primary prostate cancer specimens. Small interfering RNA-induced reduction of ASAP1 protein expression markedly suppressed in vitro PC-3 cell migration (approximately 50%) and Matrigel invasion (approximately 67%). This study suggests that the ASAP1 gene plays a role in prostate cancer metastasis and may represent a therapeutic target and/or biomarker for metastatic disease.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl‐2 and Bcl‐xL genes

To determine whether a specifically designed bispecific (Bcl‐2/Bcl‐xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl‐2 and Bcl‐xL are both anti‐apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer.