Low molecular weight polycationic polyamines are found in cells in millimolar concentrations and are essential for mammalian cell proliferation, survival and function [1]. Polyamines are associated with nucleic acid metabolism, maintenance of chromatin structure, regulation of specific gene expression, ion channel modulation and membrane stability [1]. The synthesis and catabolism of the polyamines is exquisitely regulated. Polyamines and their biosynthetic enzymes are co-ordinately regulated with growth controls, and polyamine dysregulation frequently occurs in cancer [2]. Thus, targeting this pathway may provide therapeutic advantage in cancer and other hyperproliferative diseases. The polyamine pathway is a downstream target of known oncogenes and the inhibition of polyamine synthesis disrupts the action of these genes [2]. It also appears that the polyamines are critical to the activity of a number of histone deacetylase inhibitors [3]. Thus, the polyamine pathway is a site of therapeutic intervention that is common to, and distal to, a number of validated targets and drugs that interfere with polyamine metabolism and function should have utility both alone and in combination with other agents.

Several approaches to modulating the polyamine pathway have been utilized to date [2]. Targeting the key biosynthetic enzymes has yielded important scientific tools and some clinical success. Difluoromethylornithine, an inhibitor of the first enzyme in the mammalian polyamine biosynthetic pathway, ornithine decarboxylase, is approved for use in trypanosomiasis and has shown promise in the therapy of brain tumours [4]. It has also displayed excellent activity as a chemoprevention for colon polyps, when used in combination with sulindac [5]. However, enzyme inhibitors of the polyamine pathway have generally had limited clinical success to date. A second approach uses dysfunctional synthetic polyamine analogues to competitively inhibit natural polyamine functions. Many of the analogues synthesized to date take advantage of the self-regulatory properties of the natural polyamines and significantly modulate the activity of the biosynthetic and catabolic enzymes, lowering endogenous polyamine levels. Additionally, some of these compounds may function as polyamine mimetics, binding to normal polyamine binding sites in the cell to attenuate normal functions [6].

The compound utilized in this study, PG-11047, is a second generation polyamine analogue of N ¹, N ¹²-bisethylspermine (BESpm) [7]. PG-11047 is a conformationally restricted analogue of BESpm with a cis double bond between the central carbons. It is thought to inhibit proliferation and other cellular functions by competing with natural polyamines. It is active against human cancer cells both in vitro [7] and in mouse xenografts [8]. It has shown positive results in the treatment of lymphoma in a phase I human clinical trial as well as in a phase II study for prostate cancer. It has thus far displayed a very benign toxicity profile and a phase I monotherapy trial is nearing completion [2]. It is also in a phase 1b trial in combination with each of seven different approved drugs (Clinical Trial Identifier: NCT00705874). For the combination of PG-11047 with docetaxel or gemcitabine maximally tolerated doses have been reached. Dose escalation of PG-11047 in combination with either bevacizumab, erlotinib, cisplatin, 5-fluorouracil or sunitinib malate is ongoing. With phase II trials emerging as the next step in the evaluation of the efficacy of PG-11047, specific pre-clinical studies are being undertaken to help guide the choice of appropriate clinical subjects. Such preclinical studies include extensive in vitro screens in order to provide an insight into the subpopulations of specific cancer types that might be amenable to treatment with PG-11047, while at the same time identifying putative molecular markers for the prediction of sensitivity or resistance to treatment.

Breast cancers develop through multiple genetic and epigenetic changes resulting in a wide range of cancer phenotypes. The resulting breast cancers have been classified, based on their gene expression profiles, into five distinct subtypes that are associated with diverse tumour characteristics and clinical outcome [9, 10] and into three groups based on their genomic characteristics [11]. Treatment of a limited number of human breast cancer cell lines in culture with PG-11047 has been reported to inhibit cell growth and/or induce cell death [12, 13]. To better delineate the response of breast cancer cells to PG-11047, we studied the antiproliferative activity of this drug in a panel of 48 breast cell lines (including six non-malignant breast cell lines) that mirror many transcriptional and genomic features present in primary human breast tumours [14]. The initial characterization of this panel shows that the cell lines are comprised of three subtypes designated basal A, basal B and luminal. The basal A subtype corresponds most closely to tumours designated as basal-like [15, 16] by expression profiling while the basal B subtype corresponds most closely to tumours designated as claudin-low [17] (and Spellman et al., personal communication). The basal A and basal B subtypes will be referred to as basal and claudin-low, respectively, in the following text. Our studies show that basal subtype breast cancer cell lines are most sensitive to treatment with PG-11047 based on the dose required to inhibit 50% relative growth (GI₅₀). Correlations between cell line responses quantified by the GI₅₀ and the transcriptional and genomic characteristics of these cell lines identified a set of 250 genes that are associated with response. A signature comprised of 13 of these genes is proposed as a predictor of response to PG-11047 treatment in breast tumours.

Polyamines are required for cellular viability and elevated levels are found in many tumour types, including breast cancer [2], making polyamine synthesis an attractive target for chemotherapy. PG-11047 is a second generation polyamine analogue specifically designed as a therapeutic agent [7]. While it is reported to effectively inhibit cell growth in lung, breast and colon cancer cell lines [7, 12, 24], only a limited number of cell lines had previously been studied. Since breast cancer is now known to be comprised of multiple genomic and transcriptional subsets [10, 14] that progress and respond to therapy differently, we analysed quantitative responses to PG-11047 in a collection of 42 breast cancer and six non-malignant breast cell lines in order to identify biological and molecular features associated with response. Figure 1 shows that the basal subtype is most strongly inhibited by treatment with PG-11047, based on GI₅₀ response profiles. Basal subtype breast cell lines mirror many molecular features of basal-like primary breast tumours [15, 16] including low expression of oestrogen receptor and ERBB2 and high expression of keratin 5/6/14 and EGFR (Spellman et al., personal communication). This suggests that, PG-11047 may be preferentially effective against this more aggressive breast cancer subtype. This is consistent with observations made in a study of the polyamine analogue, N ¹, N ¹¹-diethylnorspermine (DENSPM) [25]. An analysis of BrdUrd incorporation and apoptosis induction in sensitive and resistant cell lines suggests that responses to micromolar concentrations of PG-11047 mainly involve reduced cell cycle traverse rather than induction of apoptosis. Holst et al. [13] also reported inhibition of growth in the breast cancer cell lines with PG-11047, although they observed a stronger apoptotic response. This discrepancy might be explained by differences in assay conditions, since they assessed apoptosis as an increase in the fraction of cells showing sub-G1 DNA content, while our study was based on analysis of apoptosis related caspases

The concentration of drug needed to inhibit growth measured as GI₅₀ or TGI ranged over four orders of magnitude which meant that molecular features associated with response could be identified with confidence. The correlation of transcription profiles of the cell lines with their GI₅₀ sensitivity identified 250 genes whose expression levels were associated with response to PG-11047. Network and pathway analyses of these genes are summarized in Figure 2 and Table 1. Increased interferon signaling was implicated with increased sensitivity to PG-11047 by both pathway and network analyses (Network 2). This is consistent with the observation that interferon inhibits the activity of ornithine decarboxylase (ODC) [26]. We speculate that cells with high interferon activity are preferentially sensitive to PG-11047 because interferon-induced down regulation of ODC reduces the endogenous pool of the polyamines with which PG-11047 must compete to affect its inhibitory functions. Other signalling pathways and networks implicated in Table 1 and Figure 2 have been reported to be differentially active in basal and luminal tumours and cell lines. For example, pathway activities associated with basal subtype tumours involve Ephrin receptor [14, 27], BRCA1 [28] and integrins [29]. The strong basal subtype specificity of PG-11047 probably explains the associations with these pathway activities. Differential sensitivity of basal and luminal cells to PG-11047 also probably explains the structure of Network 1 in Figure 2, since this network included several genes with strong subtype specific expression [14].

A further analysis of the 250 genes associated with response to PG-11047 identified 13 genes whose levels were strongly and independently associated with response (Table 2). We propose that a clinical response predictor could be generated by assaying the levels of expression of these genes. To this end, we evaluated the applicability of the 13 gene set in stratifying breast tumour transcription datasets. We used the model to predict the sensitivities of the tumour samples in the Chin et al. [11] tumour panel (total = 118 samples). We found that there is a tendency toward separation between the predicted sensitivities of basal versus non-basal tumours (P = 0.08). The stratification improved if we restricted the analysis to high confidence predictions of the model (89/118 samples): P = 0.01. This analysis supports the utility of PG-11047 in the treatment of basal-subtype tumours.

Assessments of the contributions of these 13 response-associated-genes to breast cancer pathophysiology may provide insights into breast cancer responses to PG-11047 that were not directly tested in this study. Table 2 shows that increased sensitivity to PG-11047 is associated with lower expression of WASL, CST3, DEAF1 and ACSL3 and higher expression of GCLM, LAMA3, SSRP1, ACYP1, CYLD, PRPF18, AMFR, PPP1R2 and LOH11CR2A.

Several of these associations suggest that PG-11047 may act to inhibit aspects of migration/metastasis. For example, the protein encoded by CST3 is an antagonist of TGF-β signaling and has been shown to be significantly down regulated in many breast cancers [30]. Thus, PG-11047 may be preferentially effective in cells with active TGF-β signaling, a phenotype that has been associated with aggressive cancer behavior including increased migration/metastasis [31] Reduced expression of WASL and increased expression of AMFR also have been reported in breast cancer tissue [32, 33] and associated with cellular migration [33–35]. Increased AMFR expression also is associated with increased phospho-AKT levels in primary human breast cancers [36]. Consistent with this, we observed that a higher protein level of phospho-AKT is a significant predictor for PG-11047 sensitive cells (Additional File 4). The implication of PG-11047 as an inhibitor of motility/metastasis is further supported by the fact that aspects of motility are known to be regulated by polyamines [37]. The associations of gains of the chromosomal regions near AMFR (16q21) and loss near CST3 (20p11.2) suggests that the associations of gene expression changes with motility/metastasis and that response to PG-11047 may be driven by genomic abnormalities.

Gene function assessments also suggest that PG-11047 may moderate aspects of epithelial to mesenchymal transition that are associated with aggressive clinical behaviour. For example, Carpenter et al. [38] have recently shown that laminin 5 may contribute to increased tumour aggressiveness resulting from epithelial to mesenchymal transition (EMT) in breast tumours. Increased expression of LAMA3, a subunit of laminin 5, is associated with response to PG-11047 suggesting the possibility that tumours responding to the drug also may experience reduced EMT. Among the other genes associated with response, GCLM and SSRP1 have been implicated in stress response [39, 40], PRPF18 and SSRP1 are involved with transcription/translation [41, 42], CYLD and PPP1R2 are involved with cell cycle regulation [43, 44] and CYLD and LOH11CR2A have been reported as anti-oncogene/tumour suppressor genes [45, 46].

We assessed the quantitative responses of 48 breast cell lines to PG-11047 and demonstrated that there is a wide range of response to treatment. Our studies suggest that basal-subtype breast cancers are preferentially sensitive to the drug and that response at lower treatment concentrations is cytostatic rather than apoptotic. Analysis of molecular features associated with response suggests that PG-11047 may also reduce metastasis-related motility and suppress the EMT phenotype. We have generated a 13 gene set response signature to identify tumours expected to respond best to PG-11047 that may be useful in the selection of patients for further evaluation of PG-11047.