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Making a Difference Targeted Accell siRNA to KLF6-SV1 affects tumor progression in a mouse model of ovarian cancer Kirsteen MacLean, Ph.D. Ovarian Carcinoma is the eighth most common cancer and the fifth leading cause of all cancer among females in the United States. Ovarian cancer now causes more deaths than any other type of cancer of the female reproductive tract, with mortality rates unchanged since the early 1980’s. Current treatment regimes rely on cytoreductive surgery using a combination of platinum and taxane-based chemotherapy administered after surgery. However, several factors make ovarian cancer a particularly difficult disease to treat successfully as presenting patients are often diagnosed after the cancer has already metastasized [1]. Numerous genetic and epigenetic changes are also evident, importantly, mutation and/or loss of tumor suppressor TP53 function (as well as other known tumor suppressor genes) have been found to be one of the most frequent genetic abnormalities associated with ovarian cancer in 60-80% of both sporadic and familial cases. Further, at least 15 oncogenes such as Myc have now been implicated in the pathogenesis of the disease, with 11 of these oncogenes showing genomic amplification. It is now estimated that at least seven signaling pathways are activated in over 50% of cases of ovarian cancer giving rise to a plethora of mutations ultimately affecting the balance of cellular proliferation, apoptosis and autophagy [2]. Work by John Martignetti’s group has previously demonstrated that decreased levels of the Kruppel-like zinc transcription factor (KLF6), a known tumor suppressor, and increased levels of its alternatively spliced oncogenic isoform, KLF6-SV1, have been linked to ovarian cancer progression and poor prognosis. Indeed reduction of KLF6-SV1 by targeted small interfering RNA (siRNA) approaches was found to dramatically affect cell proliferation, invasion, colony formation and angiogenesis in various cancer cell lines belying the importance of this sliced variant to tumor progression [3]. Given the challenges associated with current ovarian cancer treatment, including recurrence and chemoresistance, these researchers investigated the molecular basis of the up-regulation of KLF6-SV1 in ovarian cancer and potential therapeutic value of systemic administration of a novel chemically modified KLF6-SV1 siRNA molecule (Accell siRNA targeted to KLF6-SV1) to a more clinically relevant xenograft mouse model of disseminated ovarian cancer [4]. Interestingly, KLF6-SV1 inhibition by Accell siRNA (siSV1) was found to dramatically increase survival in mice bearing intraperitoneal (i.p) ovarian cell tumors. Further the group was able to demonstrate that siSV1 in combination with the routinely used platinum-based cisplatin treatment regimen synergistically inhibited tumor growth and increased survival when compared against cisplatin alone (Figure 1) [5].  Figure 1. KLF6-SV1 inhibition increases survival in mice bearing i.p. tumors In vivo whole-body bioluminescence imaging of a subset of mice before (day 7) and after treatment (day 28) with either siNTC (3 mg/kg) plus cisplatin (5 mg/kg), siSV1 (3mg/kg) plus cisplatin (5 mg/kg),or cisplatin (5 mg/kg) alone. Failure of cells to engage in the apoptotic death program is a common characteristic of cancer cells and cancer progression. Links with cancer onset, chemoresistance and apoptosis were first recognized with the discovery of the B-cell lymphoma gene gene-2 (Bcl-2 gene) as the gene activated by the t(14;18)(q32;q21) translocation with immunoglobulin regulatory loci in 85-90% of follicular lymphoma. It is now clear that deregulated Bcl-2 is a hallmark of many cancers including ovarian cancer. Homologues of Bcl-2 have now been identified and are defined by the presence of conserved motifs known as the Bcl-2 homology domains (BH1-4). To this end there are now three well described families of Bcl-2 members: the anti-apoptotic (including, Bcl-2, Bcl-XL and Mcl-1); the multi-domain pro-apoptotic (Bax and Bak) and the BH3-only (including Puma, Noxa, Bim and Bad). There are two major pathways by which cells can then commit suicide: intrinsic signals mediated by regulators from within the cell that includes cytochrome c and downstream effects on the Bcl-2 family and extrinsic signals provoked by the engagement of the Fas family of death receptors. The key effectors of both these pathways are a group of conserved proteases termed caspases (cysteine-directed aspartate specific proteases) which recognize and cleave substrates required for cellular integrity [6]. A role for KLF6-SV1 in regulating apoptosis was previously suggested for other cancer models such as liver and lung; however the underlying mechanisms for this were not explored. In ovarian cancer cell lines, inhibition of KLF6-SV1 by siRNA was found to not only increase apoptosis but to also induce caspase cleavage. To examine the molecular mechanisms further the Martignetti group investigated several Bcl-2 family genes following KLF6-SV1 inhibition, including members of the anti-apoptotic Bcl-2 family (Bcl-2 and Mcl-1), the BH3-only proteins (PUMA, Bad, NOXA), and the multi-domain pro-apoptotic protein BAX [5]. Among these members, relative to other Bcl-2 family members, NOXA, which can be induced both by p53-dependent or p53-independent stimuli, was significantly up-regulated following siSV1 treatment. Indeed, when siSV1 and cisplatin were combined, NOXA mRNA was induced seven-fold. Most notably, the effects of NOXA manifested in the treated ovarian cell lines were also observed in the tumors harvested from siRNA-treated mice. siSV1–mediated apoptosis could be completely abrogated following NOXA silencing demonstrating the importance of NOXA levels in determining the apoptotic phenotype associated with inhibition of KLF6-SV1. The ratio of the pro-apoptotic to anti-apoptotic Bcl-2 family members ultimately determines a threshold for induction of the cell death pathway. NOXA binds Mcl-1 and antagonizes its anti-apoptotic function Therefore; the group examined the indirect effects that KLF6-SV1 might have on Mcl-1 protein levels. Following cisplatin-induced treatment, Mcl-1 protein is rapidly degraded by the proteasome, and in turn, NOXA is released and induces apoptosis [7]. In the Martignetti study, Mcl-1 evaded complete degradation in ovarian cancer cells overexpressing KLF6-SV1 despite cisplatin treatment. Conversely, targeted inhibition of KLF6-SV1 resulted in decreased endogenous Mcl-1 levels, further supporting a regulatory effect of KLF6-SV1 on Mcl-1 levels [5]. Mechanistic insight now provided by Martignetti and co-workers using a chemically modified siRNA to allow in vivo delivery has established KLF6-SV1 as being a novel anti-apoptotic protein, importantly, functioning independent of a tumor cells p53 status, which suggests its potential as a novel target for anticancer therapy either as a single agent or combined with the current gold standard platinum-based drugs. Further, the novel experimental siRNA methodologies presented herein certainly highlight the utility of RNAi approaches as an adjunct to standard therapeutic regimens for ovarian cancer. Perhaps therefore we should also consider effective treatments to focus on the design of chemical modifications to increase siRNA stability, tumor uptake and prolonged duration of target silencing? References
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