Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY USA 14263
Tel: 716 - 845 - 3002
Fax: 716 - 845 - 8857
E-mail: carl.porter@roswellpark.org

Program
Drug Discovery, Targeting Polyamines

The overall research goals of this laboratory are (a) to identify new regulatory events involved in cancer cell proliferation and apoptosis, (b) to validate their potential as anticancer targets; (c) to identify and develop strategies or therapies directed towards those targets and (d) to utilize such strategies to investigate regulatory mechanisms related to the initiation and control of neoplastic cell growth.  At present, these efforts focus on polyamines, novel small cationic molecules increased in the early stages of tumor cell growth and known to be essential for initiating and sustaining that process.  The suitability of these various effectors as target sites in cancer chemotherapy is indicated by the association of polyamines with neoplastic cell growth and by the fact that interference with polyamine biosynthesis and/or function inhibits cell growth.  In contrast to the more traditional approach of inhibiting polyamine biosynthetic enzymes, we have developed an anti-proliferative approach in which polyamine analogs are used to regulate key enzymes and related proteins.  By suppressing biosynthesis and turning on catabolism, the polyamine analog, N1,N12-diethylnorspermine (DENSPM) achieves rapid polyamine depletion to a degree not attainable with inhibitors or combinations of inhibitors. The net result is the elimination of natural polyamine pools and their replacement with an analog incapable of substituting for polyamines in growth-related functions.  Through studies involving this laboratory, two representative polyamine analogs and inhibitors have attained Phase II clinical evaluation against solid tumors and lymphomas. We emphasize that our pharmacological and genetic investigations span a range of systems involving cell culture, molecular biology, mouse model systems and patient material. 

Progress

In certain cells such as SK-MEL-28 human melanoma cells, DENSPM induction of the polyamine catabolic enzyme spermidine/spermine N1-acetylatransferase (SSAT) is associated with rapid apoptosis.  Because DENSPM down-regulates polyamine biosynthesis and potently up-regulates the SSAT, we used small interfering RNA duplex (siRNA) to determine the role of SSAT induction in mediating apoptosis (Chen et al. Oncogene 2003). siRNA duplexes were targeted to three independent sites in the SSAT mRNA coding region (siSSAT).  When transfected under nontoxic conditions (83 hM), two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 human melanoma cells.  Treatment of SK-MEL-28 cells with 10 mM DENSPM in the presence or absence of 83 hM siSSAT selectively prevented a 1400-fold induction of SSAT activity by ~85% and prevented depletion of spermine (Spm) pools by ~60%. SSAT-directed siRNA also prevented DENSPM-induced caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining.  Overall, the data unequivocally link analog induction of SSAT to Spm pool depletion and to apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed at a polyamine gene and the first direct demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis.

In a subsequent study, the role of role of EGR-1 and MAPK pathways in affecting cell cycle arrest was investigated.  The lead came from a microarray analysis of DENSPM-treated cells and while EGR-1 was not found to affect cellular outcomes the related MAPK pathway did.  Onset of apoptosis was preceded by an intense phosphorylation of the MAP kinases including ERK1/2, JNK and p38 in both SK-MEL-28 and MALME-3M cells.  A role for oxidative events was confirmed with the polyamine oxidase inhibitor MDL-75275 and the antioxidant N-acetyl-L-cysteine which when used in combination with DENSPM, decreased MAPK activation and as previously shown, reduced apoptosis.  While having no effect in SK-MEL-28 cells, the MEK-1 inhibitor PD-98059 reduced activation of all three kinases, prevented p21waf1/cip1 induction in MALME-3M cells and enhanced apoptosis as indicated by accelerated caspase-3 activation and annexin V staining.  The generality of this effect was demonstrated in additional melanoma lines. Thus, MAPK activation appears to contribute to the p53 initiated induction of p21waf1/cip1 (23) by prolonging and intensifying G1 arrest and thereby delaying apoptosis.  The importance of the MAP kinase pathways (in particular ERK1/2) in determining the biological response to DENSPM treatment is clearly dependent on the genetic environment of the cells and it appears that p53 may play a role in this regard.  It is possible that the MAP kinase inhibitors now under clinical development may be used to enhance melanoma responsiveness to DENSPM providing, of course, that such agents do not similarly enhance host toxicities.   Published as Chen et al., Cancer Research, 2003.

In a related study, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs) at the level of caspase 3.   Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP (livin) protein (but not XIAP) levels by 18 h via a Smac-independent pathway.  Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome.  The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interference RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment.  Among the melanoma cells lines, survivin degradation correlated temporally with the onset of DENSPM-induced apoptosis or growth inhibition.  Merging these present findings with prior findings gives rise to the following sequence of events in DENSPM-treated SK-MEL-28 cells.  DENSPM activates polyamine homeostatic mechanisms leading to down-regulation of key biosynthetic enzymes and potent up-regulation of SSAT.  The latter results in rapid depletion of polyamine pools, possible interference with polyamine function (due to acetylation) and/or to liberation of oxidative products via SSAT-coupled oxidases.  One or more of these events then initiate apoptotic signaling. Downstream pro-apoptotic effectors include release of cytochrome c and Smac/Diablo from mitochondria, loss of survivin and ML-IAP levels and activation of caspases-9 and -3.  In the absence of the moderating influences of survivin and ML-IAP, activated caspases accelerate apoptosis.   Published as Chen et al., Oncogene, 2003.

Recognizing the importance of SSAT as a major polyamine enzyme and as a pharmacological determinant with clear antiproliferative potential, we sought to identify downstream polyamine oxidase (PAO) due to its potential for liberating oxidatively disruptive by-products.  In the course of these studies, we discovered a new enzyme--an oxidase that preferentially cleaves Spm in the absence of prior acetylation (unlike PAO). A BLAST search using maize polyamine oxidase sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma—the encoded proteins differed by 20 amino acids.  When either cDNA was transiently transfected into 293 cells, intracellular Spm pools decreased by 75% while Spd and N1-acetylspermidine pools increased suggesting that Spm was selectively and directly oxidized by the enzyme and that it lacked the expected preference for acetylated polyamines.  Substrate specificity using lysates of oxidase-transfected 293 cells confirmed that the newly identified oxidase strongly favored Spm over N1-acetylSpm and that it failed to act upon N1-acetylSpm, Spd or the preferred PAO substrate, N1, N12-diacetylSpm.  The enzyme was inducible by polyamine analogs at the level of mRNA.  Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated SMO in order to distinguish it from the PAO involved in polyamine back-conversion.    Published as Vujcic et al. (2002).

In a renewed search of the real PAO, we again applied a BLAST search strategy using SMO sequences and identified novel oxidase sequences located on human chromosome 10 and mouse chromosome 7.  Homologous mammalian cDNAs derived from human brain and mouse mammary tumor were deduced to encode ~55 kDa proteins having 82% sequence identity.  When either cDNA was transiently transfected into 293 cells, intracellular Spm pools decreased by ~30% while Spd increased 2- to 4-fold.  Substrate specificity determinations with the lysate assay revealed a preference ranking identical to that reported for purified PAO and distinctly different from the recently identified SMO.  Monoethyl- and diethyl Spm analogs also served as substrates for PAO and were internally cleaved adjacent to a secondary amine.  We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway.  By Northern blot, PAO mRNA was much less abundant in 293 cells than SMO or SSAT mRNA and all three were differentially induced in a similar manner by selected polyamine analogs.  This identification of PAO sequences together with the recently identified SMO sequences provides new opportunities for understanding the dynamics of polyamine homeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogs and inhibitors.   Published as Vujcic et al. Biochem J, 2003.

In search of additional polyamine-directed acetyltransferases, we applied homology search methods and identified novel sequences belonging to a second SSAT-2 with a chromosomal location at 17p13.1, which is distinct from SSAT-1 at Xp22.  Human SSAT-2 cDNA derived from small cell lung carcinoma was deduced to encode a 170 amino acid protein having 46% sequence identity and 64% sequence similarity with SSAT-1.  When transiently transfected into HEK-293 cells, SSAT-1 decreased Spd and Spm pools by ~30% while transfected SSAT-2 had no effect on intracellular polyamine or acetylated polyamine pools. When activity was assayed on extracts from transfected cells, both SSAT-1 and SSAT-2 demonstrated much higher enzyme activity than vector-transfected cells. The data suggest that in intact cells, SSAT-2 is compartmentalized and may not have access to intracellular polyamines or to the enzyme cofactor, acetyl-CoA.  By substituting candidate substrates in the enzyme assay, we determined that SSAT-1 prefers Spd >> Spm > AcSpm while SSAT-2 prefers Spm = Spd >> AcSpm.  While SSAT-1 mRNA was inducible by polyamine analogues in a variety of cell lines, SSAT-2 was not.  In collaboration with Dr. Pendyala, we recently found that, like SSAT-1, it is highly induced by platinum drugs (unpublished).  The existence of an active but possibly sequestered SSAT-2 enzyme suggests that, under certain conditions (i.e. possibly platinum drug cytotoxicity), it may be recruited into metabolic activity.    Published as Chen et al. Biochem J, 2003.

Translational Studies:  In in vivo studies, we continue to work with structurally constrained polyamine analog synthesized and provided by S’LIL Pharmaceuticals (Madison, WI) exhibits meaningful antitumor activity against prostate and pancreatic carcinoma and prostate carcinoma xenografts when administered on a repeating dose schedule.  This work was recently published by Frydman et al. (2003) and has led to the preclinical development of this analog for clinical trials against prostate cancer.   Preclinical toxicity has revealed acceptable toxicities and the drug is moving forward.

Select Publications

• Vujcic S, Diegelman P, Bacchi CJ, Kramer DL, Porter CW. Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin. Biochem. J. 367:665-675, 2002.
• Vujcic S, Liang P, Diegelman P, Kramer DL, Porter CW.  Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion.  Biochem J.  370:19-28, 2003.
• Chen Y, Kramer DL, Li F, Porter CW.  Loss of inhibitor of apoptosis proteins as a determinant of polyamine-analogue-induced apoptosis in human melanoma cells Oncogene 22(32):4964-4972, 2003.
• Chen Y, Kramer D, Vujcic S, Jell JA, Kisiel N, Diegelman P, Porter CW. SiRNA suppression of polyamine analogue-induced spermidine spermine N1-acetyltransferase.  Mol. Pharm. 64(5):1153-1159, 2003.
• Chen Y, Vujcic S, Liang P, Diegelman P, Kramer D, Porter CW.  Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase.   Biochem J. .  Biochem J. 373(3):661-667, 2003.
• Chen Y, Alm K, Vujcic S, Kramer DL, Kee K, Diegelman P, Porter CW.  The role of Mitogen-activated protein kinase activation in determining cellular outcomes in polyamine analogue-treated human melanoma cells.  Cancer Res. 63(13):3619-3625, 2003.