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. 2026 Sep 29;14:126. doi: 10.1186/s40364-026-01007-w

A biomarker-driven approach to targeting polyamine depletion in triple negative breast cancer

Chloe A K White 1, Janice C Wu 1, Thomas J Velenosi 1,✉
PMCID: PMC13625368  PMID: 42811374

Abstract

Background

Triple negative breast cancer (TNBC) is the most aggressive and heterogenous breast cancer subtype. Polyamines are essential for TNBC growth and proliferation; however, therapeutic strategies targeting polyamine metabolism have shown limited efficacy in TNBC. In this study, we demonstrate a biomarker-driven approach to treating TNBC through polyamine depletion. As the rate limiting catabolic enzyme in the polyamine pathway, spermidine/spermine N1-acetlytransferase (SAT1) reduces polyamines and generates the metabolite, diacetylspermine.

Methods

TNBC cell lines, ex vivo tumor slices, and in vivo patient-derived xenograft (PDX) models with low, moderate and high baseline levels of SAT1 were used to determine whether the efficacy of polyamine depletion therapy depends on their SAT1 metabolic phenotype.

Results

TNBC with high baseline SAT1 expression was sensitive to the SAT1 agonist N1, N11-diethylnorspermine (DENSpm) resulting in reduced cell viability, polyamine depletion and elevated diacetylspermine production. Cell viability in moderate baseline SAT1 expressing TNBC was further reduced by combining DENSpm with both therapeutic and subtherapeutic doxorubicin treatment. In vivo, the addition of DENSpm to doxorubicin treatment further attenuated tumor growth in moderate SAT1 expressing TNBC-PDX, which corresponded to a sustained increase in urine diacetylspermine as a biomarker of treatment effectiveness. Mechanism delineating experiments demonstrated that doxorubicin induces SAT1 expression while DENSpm promotes productive splicing, effectively increasing and sustaining SAT1 function, depleting polyamines and increasing diacetylspermine production.

Conclusions

In conclusion, these findings suggest that an SAT1-defined metabolic phenotype may enable a precision-guided strategy to effectively target polyamine depletion as a TNBC treatment and that treatment effectiveness may be monitored through evaluation of urine diacetylspermine as a non-invasive biomarker of treatment response.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40364-026-01007-w.

Keywords: Triple negative breast cancer, Polyamines, Chemotherapy, Metabolic phenotype, Metabolite biomarker, Precision therapy

Background

Breast cancer represents the most commonly diagnosed cancer, and leading cause of cancer-related death in women worldwide [1–3]. Triple negative breast cancer (TNBC) accounts for about 15–20% of all breast cancer diagnoses [1, 4–6]. Hormone and HER2 positive breast cancers can be treated with targeted therapies but, in the absence of drug targets, treatment options for TNBC are limited to untargeted therapy. Standard-of-care TNBC treatment consists of several untargeted chemotherapy drugs: cyclophosphamide, taxanes, and anthracyclines including doxorubicin [4, 5]. While the recent addition of pembrolizumab immunotherapy has greatly improved treatment outcomes, 35–40% of TNBC patients still do not achieve a pathological complete response, emphasizing an urgent need for more effective and safe TNBC therapeutics [7–9].

TNBC growth and proliferation is fueled by metabolic reprogramming and supported by polyamines. Putrescine, spermidine, and spermine are small cationic polyamines required for important cellular processes including replication, and are frequently elevated in cancer cells to support tumor progression [10–12]. Polyamine levels are tightly regulated by spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting catabolic enzyme that acetylates polyamines and leads to their cellular excretion [13].

Diacetylspermine, a product of SAT1 polyamine acetylation and depletion, can be reliably measured and quantified in urine. Many studies have shown that urine diacetylspermine can serve as a useful non-invasive, diagnostic and prognostic biomarker for several cancers [14–22]. Levels of urine diacetylspermine are frequently elevated in patients with malignant tumors compared to healthy individuals.

Depletion of polyamines attenuates cell replication and limits tumor growth [23]. Accordingly, SAT1 induction to deplete polyamines has been explored as a target to treat various cancers, including TNBC [24, 25]. N1,N11-diethylnorspermine (DENSpm) is a structural polyamine analogue, which acts to potently induce the activity of SAT1 and stabilize SAT1 mRNA transcripts, increasing their half-life and leading to further polyamine acetylation [26, 27]. Preclinical studies have shown that SAT1 induction using DENSpm can be an effective strategy for reducing or inhibiting tumor growth [24, 28–30]. In Phase I trials studies, DENSpm was shown to be safe [31–33]; however, when used in an untargeted Phase II clinical trial with only 16 metastatic breast cancer patients, DENSpm was not broadly effective[34]. TNBC tumors have elevated SAT1 expression compared to other breast cancer subtypes, but expression levels can vary between individual patients [14]. Therefore, when using a biomarker-guided approach, tumors with high baseline SAT1 expression may undergo further SAT1 induction and growth inhibition when treated with DENSpm. Our previous work has demonstrated that SAT1 is induced by doxorubicin treatment [14]. Combining DENSpm with doxorubicin during treatment may further increase SAT1 expression and polyamine depletion.

Polyamine analogues have been used in combination with a variety of chemotherapeutic agents in a phase I clinical trial and have been shown to be clinically safe [35]. The goal of this study is to apply a biomarker driven approach to target TNBC tumor metabolic phenotypes that may be sensitive to SAT1-mediated polyamine depletion. This may lead to the repurposing of DENSpm as an effective targeted therapeutic for a subgroup of TNBC patients, with the potential to accelerate clinical uptake.

We hypothesized that TNBC baseline SAT1 expression levels may indicate metabolic vulnerabilities to polyamine depleting therapy. Here, using a panel of TNBC cell lines and TNBC-PDX models with different baseline expression levels of SAT1, we show that high-baseline SAT1 expressing TNBC is more sensitive to DENSpm treatment and that combination doxorubicin-DENSpm treatment is more effective at inducing SAT1 expression, depleting polyamines, reducing cell proliferation, attenuating tumor growth, and increasing urine diacetylspermine compared to standard-of-care doxorubicin alone in TNBC with moderate baseline expression of SAT1.

Methods

Cell lines

The TNBC cell lines MDA-MB-231, BT549, and MDA-MB-468 were obtained from the National Cancer Institute Developmental Therapeutics Repository. MDA-MB-453 cells were obtained from ATCC. All cell lines were maintained in in RPMI containing L-glutamine (Corning, cat no. 10-040-CV), 10% FBS (Fisher Scientific, cat no. FB12999102), and antibiotic-antimycotic solution (Corning, cat no. 30-004CI).

Animal models

TNBC patient-derived xenograft (PDX) models were generated using TM00096 (TM96), TM00098 (TM98), and TM00099 (TM99) tumors obtained from Jackson Laboratories. PDX tumors were engrafted subcutaneously onto the right flank of NSG (Strain No. 005557) mice for all experiments. All animal studies were approved by the University of British Columbia Animal Care Committee. The maximum tumor burden permitted by the approved animal protocol was 2000 mm3 and was not exceeded in this study.

Ex vivo tissue slice experiments

Untreated NSG mice bearing TNBC-PDX tumors between 1000 mm3 and 1500 mm3 were euthanized and tumors were excised aseptically, as previously described [14]. Briefly, tumors were sliced along the major axis and covered with 2% low-melting point agarose (Sigma-Aldrich, cat no. A9414) heated to ~ 40 °C. Once agarose had solidified, tumors were sliced into 500 μm wide slices using an 800 series tissue chopper (McIlwain Laboratories), washed twice with PBS (Corning, cat no. 21-040-CV), and placed into 3 mL of complete media. Following all treatments, tissue slices were washed in PBS, weighed and flash frozen for subsequent experiments.

Cell viability assays

Cells were seeded at 10,000 cells per well in 96-well plates and allowed to grow for 24 h before treatment. After treatment, cell viability was measured using the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich Solutions, cat no. 96992) and absorbance was measured at 450 nm.

RNA extraction, PCR and quantitative PCR

SAT1 mRNA expression levels were analyzed from cell lines and PDX tumor tissue. Total RNA was extracted using TRIzol reagent (Invitrogen, cat no. 15596018) according to the manufacturers protocol. cDNA was synthesized using qScript cDNA synthesis kit (Quantabio, cat no. 95047). PCRs were completed using the DreamTaq Green Master Mix (Thermo Scientific, cat no. K1081). PCR products were run on a 2% agarose gel and analyzed by densitometry. Quantitative PCR (qPCR) was carried out using PerfeCTa SYBR Green Fast Mix Low ROX (Quantabio, cat no. 84020) on a QuantStudio 7 Flex (Applied Biosystems). Human specific primers for SAT1 and GAPDH were previously generated [14, 36] (Table S1) and values are presented as relative expression calculated using the 2–∆∆Ct method.

LC-MS quantitation of polyamines

Cells were washed twice with PBS and harvested with TrypLE (Gibco, cat no. 12604-013), then counted and centrifuged at 500xg at 4 °C for 5 min and flash frozen in liquid nitrogen. Polyamine quantitation was performed as previously described [14]. Briefly, cells were diluted in 50 µL of 6% TCA containing internal standards, and ex vivo tissue slice samples were diluted in 20 µL/mg tissue of 6% TCA containing internal standards (100 nM d6-diacetlyspermine and 10 µM d8-spermine). Tissue slices were homogenized with a Bead Ruptor bead mill homogenizer (Omni Inc.) for two 30s cycles with a 10s pause. All samples were centrifuged at 15,000 x g for 10 min and 10µL of the supernatant was transferred to a new tube containing 70 µL of 100 mM Borax (pH = 10) with 1 mM ascorbic acid, 1 mM TCEP, and 100 µM NaOH. Polyamines were derivatized with 10 mM 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate (AQC, Cayman Chemical, cat no. 30877) for 10 min then the reaction was quenched for 10 min at 55 °C. Samples were injected onto a Waters Acquity BEH Phenyl-Hexyl column (2.1 × 50 mm) maintained at 40 °C with a flow rate 0.6 mL/min in an Exion LC AD. The mobile phase consisted of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) and run under the following conditions: 0–0.5 min, 0%B; 0.5–4 min, 0–15% B; 4–6 min, 15–30% B; 6–6.5 min, 30–99% B, 7.5–9 min 0% B. Eluting metabolites were measured in a Sciex 7500 using multiple reaction monitoring [14]. The total run time was 9 min.

In vivo study

Seven- to eight-week-old NSG mice were subcutaneously engrafted with ~ 2 mm3 pieces of moderate baseline SAT1 expressing PDX tumors (TM98). Tumors were allowed to grow until palpable (~ 100–200 mm3). Mice were then randomized into three groups and given saline (weekly, n = 5), doxorubicin (Accord, code 11601, DIN 02381982) (2 mg/kg, I.V., weekly n = 5) or combination doxorubicin (2 mg/kg, I.V., weekly) and DENSpm (MedChemExpress, cat no. 156886-85-0) (100 mg/kg, I.P., twice weekly, n = 5) for three weeks. A three-week treatment was performed in vivo to evaluate the effectiveness of combination doxorubicin-DENSpm treatment compared to monotherapy with doxorubicin. Tumors were measured thrice weekly using calipers, and tumor volumes were calculated using the following formula: volume = (length x width2) / 2. Urine was collected once weekly by placing mice individually into autoclaved metabolic cages inside a biosafety cabinet for 24 h. Mice were euthanized 28 days following initial treatment, blood and tumor tissue was collected at time of euthanasia. Urine and blood samples were centrifuged at 2000xg then urine and plasma were stored at -80 °C. All tissue samples were flash frozen in liquid nitrogen and stored at -80 °C until subsequent experiments.

Western blots

Cells were seeded in 6-well plates and allowed to grow for 24 h before treating for 24 h. After treatment, cells were trypsinized and centrifuged at 600xg for 5 min, washed with 1mL PBS, centrifuged and the supernatant was removed. Cells were reconstituted in 50µL RIPA buffer (Thermo Fisher, cat no. 89900) containing protease inhibitor. Cells were then vortexed and placed in a tube rotator at 4 °C for 2 h. After lysis, protein concentration was determined using a BCA assay (Thermo Fisher, cat no. 23227). A 20 µg protein mixture was made from each sample containing β-mercaptoethanol (Sigma Aldrich, cat no. M6250) and Lamelli buffer (Biorad, cat no. 1610747). Protein samples were then heated to 95 °C for 5 min and allowed to cool to room temperature before loading into 4–15% Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Biorad, cat no. 4568084) at 120 V. Protein was transferred from gels to nitrocellulose membranes using the Biorad Trans-Blot Turbo Blot Transfer System. Nitrocellulose membranes were then washed with PBS-T and blocked in 5% BSA for 1 h at room temperature. After blocking, membranes were washed three times with PBS-T and incubated in primary antibodies overnight at 4 °C, alpha tubulin (Cell signaling, cat no. 2144 S, 1:2500), SAT1 (Abcam, cat no. AB105220, 1:1000). Membranes were then washed three times with PBS-T and then incubated for 1 h at room temperature in goat anti-rabbit HRP conjugated secondary antibody (Cell signaling, cat no. 7074P2). After secondary incubation membranes were washed three times with PBS-T and ECL substrate (Biorad, cat no. 1705060 S) was added for chemiluminescence visualization. Membranes were then imaged and densitometry was performed using the Sapphire Biomolecular Imager (Azure Biosystems Inc.).

Statistical analysis

Data are presented as mean and standard error of the mean. Data were either analyzed by one-way or two-way ANOVA with repeated measures and P < 0.05 was considered statistically significant. All statistical analyses were carried out in GraphPad Prism versions 9 and 10. N refers to biological replicates for all cell lines, tissue slices and in vivo experiments.

Results

Modeling the clinical range of SAT1 baseline expression using TNBC cell lines and PDX models

According to The Cancer Genome Atlas (TCGA), breast cancer expression of SAT1 is relatively low compared to other cancer types (Fig. 1A). Within breast cancer subtypes, SAT1 expression levels are elevated in HER2 + and TNBC tumors compared to hormone receptor (HR) positive tumors (Fig. 1B) [14, 24, 37]. TCGA data also demonstrates interindividual SAT1 expression can vary across TNBC tumors (Fig. 1C). When z-scored, TNBC cell lines and the TCGA tumors demonstrate a similar spread and normal distribution in SAT1 expression (Fig. 1D). To model the clinical range of TNBC SAT1 expression in this study, we selected a panel of TNBC cell lines with low, moderate and high SAT1 expression (Fig. 1E) [38]. MDAMB231 and BT549 cells expressed SAT1 at the lower and higher end of the moderate range, respectively. SAT1 mRNA expression in MDAMB468 and MDAMB453 was 4.7-fold higher and 20-fold lower than the average of moderate SAT1 expressing TNBC cells (Fig. 1E). We also identified three TNBC-PDX models with low (TM96), moderate (TM98) and high (TM99) SAT1 expression that had a similar expression range to TNBC cell lines (Fig. 1F). Based on these findings (Fig. 1), the TNBC models were classified as metabolic phenotypes of low- (TM96, MDAMB453), moderate- (TM98, BT549, MDAMB231), and high- (TM99, MDAMB468) SAT1 expression, representing the clinical range of SAT1 expression within TNBC.

Fig. 1.

Fig. 1

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TNBC models representing baseline SAT1 expression and function. (A) Expression of SAT1 in various cancer types from The Cancer Genome Atlas (TCGA). (B) Expression of SAT1 in breast cancer subtypes from the TCGA. (C) Z-scored SAT1 expression in TNBC tumors from the TCGA. (D) Z-scored SAT1 expression in TNBC cell lines from DepMap. (E) Baseline untreated SAT1 expression of TNBC cell lines used in this study, measured via qPCR and normalized to an average of MDAMB231 and BT549 cell lines. (F) Baseline untreated SAT1 expression of fresh-frozen TNBC-PDX ex vivo tumor slices, measured via qPCR and normalized to the TM98 (moderate) TNBC tumor. N = 3. Statistical analysis carried out using ordinary one-way ANOVA, error bars indicate standard deviation, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The following cancer types from the TCGA were included (A): Pheochromocytoma and paraganglioma (PCPG), lower-grade glioma (LGG), acute myeloid leukemia (LAML), uterine carcinosarcoma (UCS), adrenocortical carcinoma (ACC), breast invasive carcinoma (BRCA), ovarian serous cystadenocarcinoma (OV), bladder urothelial carcinoma (BLCA), sarcoma (SARC), rectal adenocarcinoma (READ), esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), kidney renal clear cell carcinoma (KIRC), skin cutaneous melanoma (SKCM), uveal melanoma (UVM), glioblastoma multiforme (GBM), colon adenocarcinoma (COAD), kidney renal papillary cell carcinoma (KIRP), cholangiocarcinoma (CHOL), mesothelioma (MESO), testicular germ cell tumor (TGCT), head and neck squamous cell carcinoma (HNSC), thymoma (THYM), kidney chromophobe (KICH), liver hepatocellular carcinoma (LIHC), pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), prostate adenocarcinoma (PRAD), diffuse large B-cell lymphoma (DLBC), uterine corpus endometrial carcinoma (UCEC), and thyroid carcinoma (THCA)

Polyamine depletion by doxorubicin and DENSpm depends on baseline SAT1 expression

To evaluate whether SAT1 induction and subsequent depletion of polyamines depends on SAT1 baseline expression, we treated low- (MDAMB453), moderate- (BT549, MDAMB231) and high-baseline (MDAMB468) SAT1 expressing cells with doxorubicin (1 µM), DENSpm (10 µM) or a combination of both drugs for 24 h. SAT1 expression was induced by DENSpm, but not doxorubicin treatment in MDAMB468-high SAT1 expressing cells (Fig. 2G). Accordingly, only MDAMB468-high treatments containing DENSpm resulted in polyamine depletion (Fig. 2A-C) and elevated diacetylspermine production (Fig. 2D). In moderate baseline SAT1 expressing MDAMB231 and BT549 cells, doxorubicin induced SAT1 expression and, when combined with DENSpm, increased production of diacetylspermine and depleted polyamine levels (Fig. 2). In low SAT1 expressing MDAMB453 cells, only the combination of doxorubicin and DENSpm significantly induced SAT1 expression (Fig. 2G) and increased diacetylspermine production (Fig. 2D). Media diacetylspermine concentrations were below the limit of quantification in TNBC cells with moderate (BT549 and MDAMB231) and low (MDAMB453) SAT1 expression (data not shown). Therefore, N1-acetylspermidine was measured in media as a surrogate marker of acetylated polyamine excretion. Extracellular N1-acetylspermidine levels were consistent with intracellular diacetylspermine production (Fig. 2E). In high- (MDAMB468) baseline SAT1 expressing cells, SAT1 protein levels significantly increased in DENSpm, and combination doxorubicin-DENSpm treated samples (Fig. 2F). In high- (MDAMB468) and moderate- (BT549) baseline SAT1 expressing cells, treatment with 1µM doxorubicin, and 1µM doxorubicin in combination with 10µM DENSpm resulted in significantly decreased expression of peroxisomal N1-acetyl-spermine/spermidine oxidase (PAOX), further supporting the elevated levels of acetylated polyamines (Fig. 2H). These data suggest that the capacity for doxorubicin and DENSpm to induce SAT1 expression and diacetylspermine production, while depleting polyamines, depends on inherent expression levels of SAT1 at baseline.

Fig. 2.

Fig. 2

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Doxorubicin-DENSpm treatment mediated polyamine depletion depends on baseline SAT1 expression. Intracellular levels of putrescine (A), spermidine (B) and spermine (C) in TNBC cells. (D) Intracellular levels of diacetylspermine from TNBC cells. (E) Extracellular levels of N1-Acetylspermidine from TNBC cells. (F) Relative SAT1 protein levels from TNBC cells, measured via Western blot and analyzed using densitometry. (G) Relative SAT1 mRNA expression levels from TNBC cells, measured via qPCR and normalized to an average of the BT549 and MDAMB231 controls. (H) Relative PAOX mRNA expression levels from TNBC cells, measured via qPCR and normalized to controls of each cell lines. All cells were treated for 24 h with vehicle control, 1µM doxorubicin (Dox), 10µM DENSpm, or Dox + DENSpm. All polyamine levels were measured via LC/MS (A-E). Diacetylspermine data points below the limit of quantification (LOQ) were calculated as LOQ/2. Statistical analysis carried out by ordinary one-way ANOVA within each cell line. Error bars indicate standard error of the mean, N = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

DENSpm reduces TNBC cell viability when combined with subtherapeutic concentrations of doxorubicin

Standard of care TNBC treatment with doxorubicin has a maximum lifetime dose due to dose limiting cardiotoxicity [39]. Combination treatments may offer a potential strategy to lower toxic doses of doxorubicin while maintaining efficacy. All TNBC cell lines demonstrated a similar sensitivity to doxorubicin when treated with a clinically relevant 1µM dose [40], regardless of baseline SAT1 expression (Fig. 3A). To determine whether combined treatment of DENSpm with a subtherapeutic dose of doxorubicin reduced cell viability depending on baseline SAT1 expression, we treated TNBC cells with 0.1µM doxorubicin, 10µM DENSpm or a combination of both drugs. DENSpm monotreatment significantly reduced viability only in TNBC cells with higher baseline SAT1-expression (MDAMB468, BT549) but did not significantly affect TNBC cells with lower baseline SAT1 expression (MDAMB231, MDAMB453) (Fig. 3B, P < 0.05). In BT549 cells, which express SAT1 at the higher end of the moderate range (Fig. 1E), DENSpm reduced cell viability alone and in combination with subtherapeutic doxorubicin treatment. In MDAMB231 moderate SAT1 expressing cells, there was a trend towards decreased viability following DENSpm treatment, with a significant reduction in cell viability when combined with subtherapeutic doxorubicin treatment. (Fig. 3B). DENSpm did not affect cell viability in low SAT1 expressing cells (Fig. 3B). Combined DENSpm and subtherapeutic doxorubicin treatment significantly increased SAT1 expression, reduced polyamine levels, and increased diacetylspermine production in all cell lines except for MDAMB453 low-SAT1 expressing cells, where diacetylspermine levels were unchanged (Fig. 3C-G, P < 0.05). These data show that the addition of DENSpm to subtherapeutic concentrations of doxorubicin reduces cell viability and that the effect on polyamine levels and diacetylspermine production is dependent on baseline-SAT1 expression.

Fig. 3.

Fig. 3

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DENSpm reduces TNBC cell viability when combined with subtherapeutic concentrations of doxorubicin. (A) TNBC cell viability following 48 h treatment with vehicle control or therapeutic 1µM doxorubicin. (B) TNBC cell viability following 48 h treatment with vehicle control, 0.1µM Dox, 10µM DENSpm, or 0.1µM Dox + 10µM DENSpm. (C) Relative SAT1 mRNA expression of TNBC cells, measured via qPCR and normalized to an average of the MDAMB231 and BT549 controls. (D) Intracellular levels of putrescine from TNBC cells. (E) Intracellular levels of spermidine from TNBC cells. (F) Intracellular levels of spermine from TNBC cells. (G) Intracellular levels of diacetylspermine from TNBC cells. All cells used for SAT1 expression and polyamine measurement (C-G) treated for 24 h with vehicle control, 0.1µM Dox, 10µM DENSpm, or 0.1µM Dox + 10µM DENSpm. N = 3. All polyamines were measured via LC/MS (D-G). Data points below the limit of quantification (LOQ) were calculated as LOQ/2. Statistical analysis carried out by t-tests (A), or one-way ANOVA within each cell line (B-G). Error bars indicate standard error of the mean *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Combination treatment more effectively depletes polyamines and attenuates tumor growth in moderate SAT1 expressing TNBC

To determine whether doxorubicin and DENSpm combination treatment effectively deplete polyamines in TNBC tumors, we generated ex vivo tumor slices using low (TM96), moderate (TM98), and high (TM99) baseline SAT1 expressing TNBC-PDX models. Spermidine is more responsive to changes in polyamine metabolism within ex vivo tumor slices than spermine [14]. DENSpm decreased putrescine and spermidine levels in high-SAT1 expressing ex vivo tumor slices; however, doxorubicin had no effect on spermidine when used alone or in combination with DENSpm (Fig. 4A, Supplemental Fig. 1A and 1B, Supplemental Fig. 2A). DENSpm also reduced spermidine levels in moderate (TM98) baseline SAT1 expressing ex vivo tumor slices and, when combined with doxorubicin, a further dose-dependent depletion occurred (Fig. 4B). In low-SAT1 expressing (TM96) ex vivo slices, DENSpm and doxorubicin had no effect on spermidine levels (Fig. 4C). While intratumoral diacetylspermine levels were not significantly altered with doxorubicin or combination doxorubicin-DENSpm in high baseline-SAT1 expressing tumors (Fig. 4D), media levels of diacetylspermine were elevated 2-fold (Fig. 4H). These data suggest that DENSpm induced SAT1 expression leading to elevated diacetylspermine production and secretion in high baseline-SAT1 expressing TNBC. Changes in putrescine were not significantly different between doxorubicin monotreatment and combination doxorubicin-DENSpm treatment in low (TM96) or moderate (TM98) models (Supplemental Fig. S3B, C), and changes in spermine levels were not significantly different in any of the three PDX models (Fig. S3D-E). Intratumoral diacetylspermine levels were significantly increased when doxorubicin was combined with DENSpm in both moderate and low-SAT1 expressing tumor slices (Fig. 4E and F). Extratumoral levels of diacetylspermine had only a slight increase above doxorubicin alone in the low-SAT1 expressing ex vivo slices at 1µM doxorubicin (Fig. 4J). Levels of extratumoral diacetylspermine were significantly elevated with combination doxorubicin-DENSpm in moderate SAT1 expressing tumors (Fig. 4I), indicating high levels of diacetylspermine tumor export. These data suggest that combination doxorubicin-DENSpm treatment is most effective at depleting polyamines and increasing diacetylspermine production in moderate baseline-SAT1 expressing TNBC tumors.

Fig. 4.

Fig. 4

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Combination DENSpm and doxorubicin treatment dose-dependently deplete spermidine levels of moderate baseline TNBC-PDX tumor slices. Intratumoral levels of spermidine from high (TM99) (A), moderate (TM98) (B), and low (TM96) (C) SAT1 expressing ex vivo tumor slices. Intratumoral levels of diacetylspermine from high (TM99) (D), moderate (TM98) (E), and low (TM96) (F) SAT1 expressing ex vivo tumor slices. Extratumoral levels of diacetylspermine from high (TM99) (G), moderate (TM98) (H), low (TM96) (I) SAT1 expressing ex vivo tumor slices. All tumor slices were treated for 24 h with a dose response treatment from 0µM to 3µM doxorubicin in the presence or absence of 10µM DENSpm. All metabolite levels were measured via LC/MS. Error bars indicate standard error of the mean, N = 3, analyzed using two-way repeated measures ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. * Indicates significant difference from vehicle control, # indicates significant differences from DENSpm treatment

The moderate SAT1 expressing models represent the majority of TNBC tumors (Fig. 1C) and demonstrated the greatest depletion of polyamines with combination doxorubicin-DENSpm treatment in ex vivo tumor slices (Fig. 4B). To determine the potential benefit of adding DENSpm to doxorubicin treatment in moderate SAT1 expressing TNBC-PDX, we treated TM98-PDX mice with vehicle, doxorubicin or combined doxorubicin and DENSpm (Fig. 5A). Combination doxorubicin-DENSpm treatment attenuated tumor growth and induced SAT1 expression significantly more than doxorubicin treatment alone (Fig. 5B and D, P < 0.05). Polyamine levels were not significantly changed except for a slight increase in putrescine levels (Supplemental Fig. 2B), but acetylated polyamine levels were significantly elevated in combination doxorubicin-DENSpm treated mice (Supplemental Fig. 2C). Notably, the combination treatment resulted in significantly elevated urine diacetylspermine that was sustained after the cessation of treatment (Fig. 5C), likely due to the DENSpm remaining in tissues at the end of the study, 10 days after the last dose (Fig. S2A). The end-of-study intratumoral tumor levels of diacetylspermine where combination doxorubicin-DENSpm treated tumors had significantly elevated levels of diacetylspermine compared to control or doxorubicin treated tumors (Fig. 5E).

Fig. 5.

Fig. 5

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In vivo tumor growth inhibition and urine diacetylspermine production were sustained for 10 days following the final combination DENSpm and doxorubicin treatment in moderate SAT1 expression TNBC-PDX. (A) In vivo study design. (B) TM98 (moderate) PDX tumor growth curve in tumor-bearing NSG mice. (C) Urine diacetylspermine levels from tumor-bearing TM98 (moderate) PDX tumors, measured via LC/MS. (D) SAT1 mRNA expression levels from TM98 (moderate) PDX tumors at end point of study, measured via qPCR and normalized to controls. (E) Intratumoral levels of diacetylspermine from TM98 (moderate) PDX tumors at end point of study, measured via LC/MS. All mice received 2 mg/kg I.V. doxorubicin (Dox) once weekly, 2 mg/kg I.V. Dox once weekly and 100 mg/kg I.P. DENSpm twice weekly or 8mL/kg I.V. vehicle control (saline) once weekly for three weeks. N = 5. Statistical analysis carried out by two-way ANOVA (B, C), or one-way ANOVA (D, E), error bars indicate standard error of the mean, * indicates difference between control and Dox + DENSpm, # indicates difference between Dox and control, † indicates difference between Dox and Dox + DENSpm. For all *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Urine diacetylspermine is a metabolic biomarker of sustained effective combination doxorubicin and DENSpm treatment

To further examine the sustained elevation of urine diacetylspermine levels observed in the in vivo study (Fig. 5C), we attempted to replicate this effect in vitro. BT549 moderate SAT1 expressing cells were treated with vehicle, doxorubicin, DENSpm or combination doxorubicin-DENSpm for 24 h, then media was removed, and cells were harvested or cultured in the absence of drug treatment for an additional 24–48 h. Doxorubicin induced SAT1 mRNA after 24 h, which was further increased by DENSpm in combined treatment (Fig. 6A). After drug removal, expression levels were maintained for 24 h, then decreased at 48 h, but remained significantly elevated. SAT1 has two major mRNA isoforms. A protein coding short isoform and a long unproductive isoform with the inclusion of exon 4 that contains several stop codons [41]. While doxorubicin induced SAT1 mRNA and diacetylspermine production, the ratio of long/short mRNA also progressively increased for 48 h after treatment was removed (Fig. 6B). Notably, DENSpm significantly reduced the ratio of long/short isoform alone and in combination treatment, effectively increasing the productive mRNA isoform.

Fig. 6.

Fig. 6

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SAT1 expression and diacetylspermine production were sustained following combination doxorubicin and DENSpm treatment. (A) Experimental design and relative SAT1 mRNA expression levels of BT549 cells, measured via qPCR and normalized to controls from each timepoint. (B) Different SAT1 isoform densitometry from BT549 cells, measured via PCR. (C) Intracellular diacetylspermine levels from BT549 cells, measured via LC/MS. (D) Extracellular diacetylspermine levels from BT549 cells, measured via LC/MS. Cell for all experiments were treated for 24 h with 1µM doxorubicin, 10µM DENSpm, or both drugs in combination, treatment was removed immediately before measurement (24T), 24 h before measurement (24T + 24U), or 48 h before measurement (24T + 48U). N = 3. Statistical analysis carried out by two-way ANOVA, error bars indicate, standard deviation (A, B), or standard error of the mean (B, C) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Similarly to mRNA expression, diacetylspermine production was induced following combined doxorubicin-DENSpm treatment, which decreased but remained significantly elevated for 48 h after treatment removal (Fig. 6C). Individual doxorubicin and DENSpm treatments also significantly increased diacetylspermine production, which was maintained for 48 h after drug removal. Extracellular diacetylspermine levels were significantly increased 24 h after treatment but did not exhibit the same pattern as intracellular levels (Fig. 6C and D). Notably, extracellular diacetylspermine levels were also maintained for 48 h after drug removal in doxorubicin and doxorubicin-DENSpm treated cells. These data suggest that doxorubicin induces SAT1 mRNA while DENSpm directs mRNA splicing towards the productive short coding SAT1 isoform leading to elevated SAT1 function and diacetylspermine production that is sustained after the cessation of treatment.

Discussion

Chemotherapy remains the most widely used treatment method for TNBC patients. Low levels of pathological complete response, high rates of drug resistance, and disease recurrence in TNBC patients emphasize the urgent need for safer and more effective therapeutic options. In addition, reliable biomarkers for monitoring disease progression and treatment response can improve outcomes [42]. Here, we conducted a study to examine the repurposing of DENSpm in combination with doxorubicin as a TNBC treatment strategy using cell lines and PDX tumors with different baseline SAT1 expression levels. We found that DENSpm and combination doxorubicin-DENSpm effectiveness was dependent on baseline SAT1 expression and function. Adding DENSpm to subtherapeutic concentrations of doxorubicin was sufficient to reduce cell viability in TNBC cell lines with high and moderate baseline SAT1 expression. Furthermore, combined doxorubicin-DENSpm treatment most effectively depleted polyamines in ex vivo tumor slices and attenuated tumor growth in moderate baseline SAT1 expressing TNBC-PDX, a metabolic phenotype that represents the majority of TNBC. Notably, effective in vivo combination treatment resulted in sustained elevation of SAT1 expression and urine diacetylspermine following treatment cessation. This result was corroborated in vitro by a persistent increase in the productive SAT1 transcript as well as intra- and extracellular diacetylspermine levels for 48 h after drug removal.

According to TCGA data, TNBC tumors exhibit higher SAT1 expression than HR positive subtypes, but within this subtype, SAT1 expression spans seven standard deviations across a normal distribution [43]. Indeed, diacetylspermine production varies substantially in TNBC cell lines [44], which is consistent with variable SAT1 expression. Historically, clinical trials have followed an untargeted approach where inclusion criteria are based on tumor site of origin and histology. Initial clinical trials using DENSpm followed this untargeted approach and failed to progress towards successful approval for cancer treatment [31–34]. Advancements in genetic sequencing and omics technologies have led to a rise in biomarker-driven clinical trials and precision medicine techniques that have improved success rates and patient outcomes [45, 46]. This study evaluated whether a biomarker-guided approach targeting specific SAT1 metabolic phenotypes could predict response to DENSpm and combined doxorubicin-DENSpm treatment.

For polyamine depleting therapy to effectively attenuate tumor growth, the rate of catabolism must exceed the rate of synthesis. This can be achieved using multiple drugs to inhibit polyamine synthesis and increase catabolism. Combining DENSpm with a methylthioadenosine phosphorylase (MTAP) inhibitor has been shown to exploit polyamine flux in prostate cancer by simultaneously depleting polyamines and inhibiting methionine salvage for polyamine production, respectively [25]. Combined polyamine synthesis inhibition also depletes polyamines in prostate cancer patients [47]. Here, we leveraged high baseline SAT1 expression as a metabolic vulnerability to promote polyamine depletion through further SAT1 induction. When high baseline SAT1 expressing TNBC cells were treated with DENSpm, intracellular spermidine was reduced to near undetectable levels, suggesting that SAT1 induction disrupts the balance between polyamine synthesis and catabolism when baseline expression is elevated. Interestingly, PAOX levels were significantly reduced by doxorubicin treatment, potentially further limiting the recycling of acetylated polyamines into the intracellular polyamine pool. Similarly, DENSpm markedly depleted spermidine in ex vivo TNBC tumor slices with high baseline SAT1 expression. Conversely, doxorubicin had no effect on spermidine depletion in high baseline SAT1 expressing TNBC cell lines or ex vivo tumor slices, indicating a consistent DENSpm-mediated polyamine depletion mechanism associated with this TNBC metabolic phenotype.

Individual doxorubicin and DENSpm treatment reduced TNBC cell viability in high SAT1 expressing cells, which was further reduced in combination treatment suggesting separate, but complementary mechanisms resulting in cell death. While doxorubicin does not induce SAT1 in high baseline SAT1 expressing TNBC, DNA damage incurred by doxorubicin combined with DENSpm mediated polyamine depletion may be an effective approach to treating the high baseline SAT1 metabolic phenotype. In low baseline SAT1-expressing ex vivo tumor slices, no significant depletion of polyamines was observed, and cell viability was not significantly affected by combination treatment compared to doxorubicin treatment alone, indicating that adding DENSpm to doxorubicin treatment may not be effective for treating tumors with a low SAT1 metabolic phenotype. These data emphasize the potential value of DENSpm in TNBC treatment when using a precision-medicine guided approach.

Moderate SAT1 expressing tumors represent the predominant phenotype in TNBC. When DENSpm was added to doxorubicin treatment in moderate SAT1 expressing tumor slices, a dose-dependent polyamine depletion occurred. In vivo, treatment of the same tumor with combination doxorubicin-DENSpm further attenuated tumor growth compared to doxorubicin suggesting that the addition of DENSpm to doxorubicin treatment may be a more effective treatment strategy compared to doxorubicin alone for the moderate SAT1 expressing phenotype.

Interestingly, SAT1 induction was sustained for 10 days following the final dose of each drug. These data suggest a persistent effect of combined DENSpm and doxorubicin treatment, long after the cessation of treatment. DENSpm has a relatively short plasma half-life but can be detected in tissue several days after dosing [48]. In this study, DENSpm could be detected in tumor, liver and kidney tissue following euthanization suggesting a long lasting effect on tumor SAT1 induction. We evaluated the potential mechanism in TNBC cell lines, which demonstrated that doxorubicin induces SAT1 mRNA expression and that DENSpm regulates alternative splicing to increase the generation of the productive short mRNA transcript. Together, these data suggest that doxorubicin and DENSpm treatment can increase long-term expression of productive SAT1 mRNA, supporting a potential sustained treatment effect in alignment with standard of care dosing schedules. Recent evidence suggests that as a polyamine analogue, DENSpm plays a larger role in metabolic shielding of phosphorylation sites that regulate alternative splicing [36]. This potential metabolic shielding effect should be further examined in future studies involving polyamine analogues. Doxorubicin has a maximum lifetime dose due to irreversible cardiotoxicity [39]. When combined with DENSpm, subtherapeutic concentrations of doxorubicin caused the greatest reduction in cell viability compared to monotreatment in moderate SAT1 expressing TNBC. These data suggest that the addition of DENSpm treatment may allow for a reduced total doxorubicin dose to mitigate future cardiotoxicity.

Although expanding treatment options for TNBC patients is important to improve TNBC survival, monitoring patient response to treatment is a useful strategy to combat drug resistance. Reliable and noninvasive biomarkers can be a valuable tool for monitoring treatment response and indicating resistance prior to clinical symptoms or radiological findings. Plasma diacetylspermine is a predictive biomarker for TNBC disease progression [44], and urine diacetylspermine is elevated in TNBC compared ER+ molecular subtypes [14]. In this study, moderate SAT1-expressing TNBC-PDX revealed significantly elevated levels of urine diacetylspermine in combination treated mice compared to controls and doxorubicin monotreatment, which was sustained for 10 days after the cessation of treatment. This result was replicated in TNBC cells where both intracellular and extracellular diacetylspermine levels remained elevated for up to 48 h after removing treatment in combination doxorubicin-DENSpm treated cells, suggesting that elevated urine diacetylspermine is tumor-derived. The sustained elevation of urine diacetylspermine may be an effective non-invasive biomarker for monitoring response to combined doxorubicin and DENSpm treatment.

Conclusions

Our study demonstrates an effective precision-guided combination treatment approach to targeting polyamine depletion in TNBC. Together, doxorubicin treatment induced SAT1 expression while DENSpm increased productive splicing, which ultimately contributed to polyamine depletion and attenuation of TNBC tumor growth. This effect was sustained long after drug treatment as indicated by elevated urine diacetylspermine. Subtherapeutic concentrations of doxorubicin effectively decreased cell viability when combined with DENSpm for moderate SAT1 expressing TNBC, which may be a potential approach to limit future cardiotoxicity. Moreover, TNBC defined by high SAT1 expression was more sensitive to DENSpm mediated polyamine depletion, suggesting a potential precision-guided therapy for this metabolic phenotype.

DENSpm is a clinically safe drug and is well tolerated as demonstrated in several Phase I clinical trials [31–33]. Although this drug has not been successful in untargeted Phase II trials [34], our results suggest that repurposing DENSpm or the use of other SAT1 agonists for the targeted treatment of specific TNBC metabolic phenotypes in combination with doxorubicin may be an effective strategy. Additionally, these results indicate the potential long-term clinical utility of urine diacetylspermine as a biomarker of treatment response in TNBC. Future studies should investigate whether repurposing of clinically safe DENSpm or other SAT1 inducing polyamine analogues [49] may more effectively achieve pathological complete response using a precision-guided approach to TNBC treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (437.9KB, pdf)

Acknowledgements

We acknowledge the assistance of Dickson Lai and the UBC Faculty of Pharmaceutical Sciences Analytical Suite for LCMS quantitation.

Abbreviations

TNBC

Triple negative breast cancer

SAT1

Spermidine/spermine N1-acetyltransferase 1

DENSpm

N1,N11-diethylnorspermine

Dox

Doxorubicin

PDX

Patient-derived xenograft

TCGA

The cancer genome atlas

MTAP

Methylthioadenosine phosphorylase

Author contributions

CAKW contributed to the conceptualization, methodology, formal analysis, investigation, and visualization. JCW contributed to the methodology and investigation. TJV contributed to the conceptualization, methodology, formal analysis, visualization, funding acquisition, and supervision. All authors contributed to the writing of the original draft as well as the editing and review.

Funding

This study was supported by the Canadian Institutes of Health Research. CAKW was supported by the Cancer Research Society (CRS) Doctoral Research Award. TJV is supported by the Michael Smith Health Research BC Scholar Award.

Data availability

The results in Fig. 1A and 1B are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. All other data are available upon request.

Declarations

Ethics approval and consent to participate

All animal studies were approved by the University of British Columbia Animal Care Committee and conducted in accordance with the guidelines of the Canadian Council on Animal Care. The maximum tumor burden permitted by the approved animal protocol was 2000 mm3 and was not exceeded in this study. All PDX models were commercially acquired from Jackson Labs and thus, a clinical research ethics protocol was not applicable for this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (437.9KB, pdf)

Data Availability Statement

The results in Fig. 1A and 1B are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. All other data are available upon request.

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