Hyperglycemic conditions proliferate triple negative breast cancer cells: role of ornithine decarboxylase

Several cancer subtypes (pancreatic, breast, liver, and colorectal) rapidly advance to higher aggressive stages in diabetes. Though hyperglycemia has been considered as a fuel for growth of cancer cells, pathways leading to this condition are still under investigation. Cellular polyamines can modulate normal and cancer cell growth, and inhibitors of polyamine synthesis have been approved for treating colon cancer, however the role of polyamines in diabetes-mediated cancer advancement is unclear as yet. We hypothesized that polyamine metabolic pathway is involved with increased proliferation of breast cancer cells under high glucose (HG) conditions. Studies were performed with varying concentrations of glucose (5–25 mM) exposure in invasive, triple negative breast cancer cells, MDA-MB-231; non-invasive, estrogen/progesterone receptor positive breast cancer cells, MCF-7; and non-tumorigenic mammary epithelial cells, MCF-10A. There was a significant increase in proliferation with HG (25 mM) at 48–72 h in both MDA-MB-231 and MCF-10A cells but no such effect was observed in MCF-7 cells. This was correlated to higher activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine synthesis pathway. Inhibitor of polyamine synthesis (difluoromethylornithine, DFMO, 5 mM) was quite effective in suppressing HG-mediated cell proliferation and ODC activity in MDA-MB-231 and MCF-10A cells. Polyamine (putrescine) levels were significantly elevated with HG treatment in MDA-MB-231 cells. HG exposure also increased the metastasis of MDA-MB-231 cells. Our cellular findings indicate that polyamine inhibition should be explored in patient population as a target for future chemotherapeutics in diabetic breast cancer.


Introduction
Diabetes and cancer have been postulated to have a correlation for the past 80 years; however, it is only within the past decade that significant epidemiological evidence has been compiled to suggest a causal link [1,2]. Strong evidence suggests that the risk for several cancer types, including cancers of the liver, pancreas, colorectum, urinary tract, breast, and female reproductive organs, is increased in diabetic patients [1,3]. Diabetes increases metastasis, recurrence, and mortality of cancer [3,4]. Currently, diabetes-cancer link has primarily been hypothesized to rely on hormonal (insulin and insulin-like growth factor 1), inflammatory, and metabolic (hyperglycemia) characteristics of diabetes [5]. Hyperglycemia promotes rapid cancer cell proliferation thus increasing cancer progression [6]. Altered glucose metabolism also contributes to rewiring of metabolic pathways for cell growth and survival [3]. Although hyperglycemia has been examined as a contributor to rapidly progressing cancer, pathways leading to this condition remain under investigation.
Studies into the molecular biology underlying breast cancer have found that polyamines, organic molecules that play a large role in eukaryotic cell growth and development, have elevated amounts in breast cancer tissue [7][8][9]. Polyamines control gene expression at transcriptional, posttranscriptional, and translational levels, modulating functions of DNA, nucleotide triphosphates, RNA, and proteins [10][11][12]. Multiple pathways regulate cellular polyamine levels, including synthesis from amino acid precursors, uptake mechanisms that utilize polyamines from diet and intestinal microorganisms, and stepwise degradation and efflux [13]. A significant body of evidence demonstrates that numerous oncogenic pathways are involved in regulating the transcription and translation of enzymes involved in polyamine metabolism, and that upregulation of polyamine biosynthetic enzymes correlates with both increased cell proliferation and tumorigenesis [14][15][16][17][18][19]. In fact, increased polyamine levels have been linked to breast, prostate, colon, and skin cancers [7,[20][21][22][23]. While normal physiological conditions maintain intracellular polyamine concentrations within narrow limits, dysregulation of polyamine metabolism can lead to various pathological conditions besides cancer, including inflammation, renal failure, stroke, and diabetes [10,24].
The polyamine pathway involves the conversion of l-ornithine (from dietary sources or conversion from l-arginine) to form putrescine (PUT), the first polyamine in the pathway [25]. This reaction is mediated by ornithine decarboxylase (ODC), a rate-limiting enzyme in the polyamine pathway, which has been shown to have increased activity in cancer ( Fig. 1) [26]. PUT is further converted to other polyamines including spermidine (SPD) and spermine (SPM) through spermidine synthase (SRM) and spermine synthase (SMS), respectively. Polyamine levels are tightly regulated by catabolism through spermine oxidase (SMOX, converts SPM to SPD), SPD SPM N 1 acetyl transferase (SAT1), and N 1 -acetylpolyamine oxidase (PAOX) [25]. A parallel pathway that feeds into the polyamine synthesis maintains intracellular levels if the primary pathway is not sufficient or is affected. This involves decarboxylation of S-adenosylmethionine (AMD1), the aminopropyl donor for synthesis of SPD and SPM.
While inhibition of polyamines through the ODC enzyme has been shown to provide protection against breast cancer [9,27], it is not known whether this would be true for diabetes-linked cancer advancement as well. In fact, polyamine pathway can be a potential target to treat chemoresistance in triple negative breast cancer [9,28]. Thus, we hypothesized that high glucose (HG) conditions advance breast cancer cell proliferation, with a possible role of polyamine pathway. Our results showed that advanced stage breast cancer cells as well as non-tumorigenic mammary epithelial cells proliferate in hyperglycemic states, and polyamine inhibition is protective in these conditions. Fig. 1 Schematic of polyamine pathway in animal cells. Primary pathway for polyamine synthesis is presented in the middle which involves the action of ornithine decarboxylase to produce the polyamines (putrescine, PUT, spermidine, SPD, and spermine, SPM) from l-ornithine. In addition, low levels of SPD or SPM also trigger input of these metabolites from a parallel alternate pathway through the decarboxylation of S-adenosylmethionine (S-AdM). Catabolism of SPD and SPM is mediated through the enzymes spermine/spermidine acetyltransferase (SSAT), and polyamine oxidase (PAOX). ODC, the first enzyme in the pathway, can be endogenously inhibited by ODC antizyme or exogenously using DFMO

Cell proliferation assay
Cells were seeded in 96-well plates at a density of 8000 cells per well and allowed to adhere for 24 h. Cells were then starved and treated (90 μl per well) with varying concentrations of glucose (5-25 mM), in the presence or absence of 5 mM DFMO. Mannitol (20 mM) with glucose (5 mM) was used as an osmotic control. After 48-72 h, cells were incubated with PrestoBlue® reagent (10 μl per well) for 10 min at 37 °C. Fluorescence was monitored at 560/590 nm (ex/ em) using a microplate reader and Gen5 software (BioTek Synergy 2; Winooski, VT, USA). Fluorescence is directly proportional to the number of living cells in each well. Blank wells included media with no cells, and this reading was subtracted from all treated wells. Percent change was calculated relative to the 5 mM glucose treatment as control.

Clonogenic assay
Cells were plated at a density of 2000 cells per well, allowed to attach overnight, and then treated with glucose and/or DFMO for 72 h. At the end of the treatment period, cells were washed with ice-cold PBS, and further incubated with complete media (no treatment) for another week, with change of media every 2 days. This is to allow establishment of colony formation from surviving cells. Colonies were washed with cold PBS, fixed with methanol for 15 min, and further stained with 0.1% crystal violet for 30 min. Plates were submerged in tap water to wash off the dye, allowed to dry overnight, and colonies with more than 50 cells were counted using a stereomicroscope. Survival fraction was calculated using the formula:

Polyamine analysis
For polyamine analysis, cells were grown and treated in T-175 flasks, trypsinized, pelleted and stored at − 80 °C until analysis. Samples were shipped on dry ice and sent to the proteomics and metabolomics facility at University of Nebraska at Lincoln for analysis. Polyamines were extracted from the cell pellets using 400 µL of chilled 5% trichloroacetic acid and after incubation on ice for 1 h with frequent vortexing. The samples were then centrifuged at 5000 rpm for 5 min at 4 °C. Supernatants were transferred to a new microfuge tube and neutralized by adding 2 M K 2 HPO 4 . The extracted polyamines were derivatized using AccQ-Tag chemistry as described previously [29]. The derivatized compounds were then analyzed using the Shimadzu Nexera II UPLC coupled to the Sciex QTRAP 6500 + mass spectrometer equipped with a TurboIon-Spray (TIS) electrospray ion source. For LC separation, a Plating Efficiency (PE) = # of colonies formed # of cells seeded × 100 Survival Fraction (SF) = # of colonies formed # of cells seeded × PE.
ACCQ-TAG ULTRA C18 1.7 μm (2.1 × 100 mm, Waters) was used flowing at 0.7 mL/min. The gradient of the mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in 100% acetonitrile) was as follow: 5% B for 2 min, to 90% B in 2.5 min, hold at 90% B for 2 min, then back to 5% B in 0.5 min. The QTRAP 6500 + mass spectrometer was tuned and calibrated according to the manufacturer's recommendations. The instrument was set-up to acquire in positive mode. Analyst software (version 1.6.3) was used to control sample acquisition and data analysis. All the metabolites were detected using MRM (Multiple Reaction Monitoring) transitions that were previously optimized using standards. For quantification, an external standard curve was prepared using a series of standard samples containing different concentrations of metabolites and fixed concentration of the internal standard.

Assay for ODC enzyme activity
For preparing samples for ODC activity assay, cells were grown and treated in T-175 flasks, and subsequently harvested in buffer containing 25 mM Tris HCl pH 7.5, 0.1 mM EDTA, and 2.5 mM DTT. Samples were stored at − 80 °C until analysis. ODC activity was determined by radioactive assay, as described previously [30], to measure the amount of 14 CO 2 liberated from l-[1-14 C]ornithine. Enzymatic activity was expressed as pmol of CO 2 /h per mg of protein.

Statistical analysis
All values obtained were expressed as mean ± SEM. Results shown were analyzed with Graph Pad software (Prism 9.0) and are representative of at least three experiments performed in replicates. Statistical comparison between more than two different groups was performed using one-way ANOVA followed by Tukey's test. Differences were considered to be statistically significant at p < 0.05.

Time and dose response assessment of cell proliferation under hyperglycemia
To assess dose response effects of glucose treatments in these cell lines, proliferation was monitored at 72 h after treatment. Both MDA-MB-231 and MCF-10A cells displayed marked increase in proliferation at 10 mM and 25 mM glucose concentrations compared to 5 mM (Fig. 2). Mannitol was used as an osmotic control, and did not cause any measurable change in proliferation compared to 5 mM glucose control. This effect was not evident in MCF-7 cells even with the highest glucose concentrations.
With respect to periods of incubation, in both MDA-MB-231 (triple negative breast cancer cell) and MCF-10A (non-tumorigenic mammary epithelial cell), HG resulted in significantly increased proliferation as early as 48 h after treatment (Fig. 3A, B) and similar trends continued 96 h post-treatments.

Effect of polyamine inhibitor DFMO on cell proliferation and colony formation
The increase in proliferation observed with HG in MDA-MB-231 and MCF-10A, was markedly suppressed with the polyamine inhibitor, DFMO, returning growth to that exhibited in LG conditions at 72 h (Fig. 4A). Addition of DFMO had negligible effects on the proliferation of both cell lines grown in LG conditions. Colony formation increased remarkably with HG treatments in MDA-MB-231 and MCF-10A cells (Fig. 4B). Interestingly, DFMO (2 mM) was effective in suppressing colonies formed for LG as well as HG treatments in all cells. Lower concentration of DFMO (2 mM) was used for these experiments due to less number of cells plated per well; higher concentration (5 mM) was cytotoxic upon longer incubation times.

Ornithine decarboxylase (ODC) activity under hyperglycemia with polyamine inhibition
Similar to proliferation and colony forming assays, ODC enzyme activity with HG exposure was elevated about two-to fivefold higher than LG in both MDA-MB-231 and MCF-10A (Fig. 5). As expected, DFMO suppressed ODC

Metastasis of late stage cancer cells in diabetes
Scratch wound healing assay was performed in late stage, metastatic, MDA-MB-231 cells. Migration of cells towards the scratch is indicative of metastasis in vivo. While wound healing was increased with incubation times, near complete healing was observed after 48 h only in HG-treated wells, indicative of metastasis ( Fig. 6; Table 1). Cells treated with LG (5 mM) showed only a partial reversal. Polyamine inhibition was ineffective in repairing the scratch under normal or HG treatments.

Polyamine levels in cells after glucose treatments
Intracellular polyamine concentrations were assayed at 48 h after treatment with glucose (5, 25 mM). PUT levels marginally increased in MDA-MB-231 cells (Table 2A) after treatment with HG but this effect was not evident in MCF-10A cells (Table 2B). SPD and SPM did not change with HG in both these cell types.

Discussion
Current evidence shows a strong link between diabetes and increased risk of several cancer types including breast, endometrial, pancreatic, hepatic, colorectal, and urinary tract [1]. Diabetes also negatively impacts cancer treatment outcomes, increasing metastasis, recurrence, and mortality of cancer [3,4]. Hyperglycemia has been considered a leading factor in promoting cancer cell proliferation [6], however controlling hyperglycemia has not shown to be effective in reducing the risk of cancer prognosis, hence other strategies to prevent and treat cancer need to be considered [1]. To our knowledge, a comparison of the effects of hyperglycemia on cell proliferation and effect of polyamine inhibition in breast cancer lines has not been performed. Therefore, the present study tested the effects of hyperglycemia on proliferation of MCF-7 and MDA-MB-231 cells (representative of low and highly invasive breast carcinomas, respectively), as well as MCF-10A cells (normal mammary epithelial cells). We also sought to elucidate the role of polyamines in diabetic cancer advancement, using a polyamine synthesis inhibitor DFMO.
Here, we have shown that MDA-MB-231 (highly invasive triple negative breast cancer) and MCF-10A (noncancerous) cells proliferate markedly after 48-72 h exposure to HG, however such effects were not observed in MCF-7 cells. Similar trends have also been reported earlier when MCF-7 cells were subjected to hyperglycemia and hyperinsulinemia, which support the differential response [31][32][33]. The varied response we observed could be attributed to the fact that MCF-7 cells are estrogen receptor positive whereas MDA-MB-231 and MCF-10A cells lack this receptor. Future studies will be performed to investigate the role of estrogen receptor in inducing proliferation of breast cancer cells under diabetic conditions. A similar study, mimicking type 2 diabetic condition in MCF-7 cells, emphasized that presence of estrogen in hyperinsulinemic states remarkably affected cell growth as compared to hyperinsulinemia alone [34].
Several cellular pathways have been investigated to understand the prognosis of breast cancer advancement in diabetes. Leptin signaling, including activation of Akt/mTOR pathway, contributes to hyperglycemia mediated increased risk of cancer in normal mammary cells, as well as cancer progression in malignant cells [32]. Proliferation, migration, and invasiveness of breast cancer cells in hyperglycemichyperinsulinemic states has also been attributed to oxidative stress which elevates urokinase plasminogen activator [35]. We tested whether polyamines are involved in this pathway, as polyamines are required for cell growth, and elevated in several cancer subytpes including skin, colon, and breast. Inhibition of the polyamine synthesis pathway, using DFMO abrogated proliferation of cells and colony formation observed with HG treatments. This was observed in both MDA-MB-231 and MCF-10A cells after 72 h treatments. Interestingly although HG did not increase proliferation To understand the role of polyamine pathway in regulating HG-mediated cell proliferation, we also measured enzyme activity of ODC, the rate-limiting enzyme involved in the polyamine pathway (Fig. 1). ODC activity was elevated considerably with HG in all cell types, and DFMO treatment was protective in reversing these effects. ODC is directly involved in the production of PUT, the first product in the polyamine biosynthetic pathway (Fig. 1). PUT levels were slightly increased with HG in MDA-MB-231 cells but did not change in MCF-10A cells. Quite remarkably, SPD and SPM concentrations did not change with HG treatments in cancer cells and normal cells. As evident from Table 2, polyamine values are markedly higher for cancer cells compared to normal cells, which is probably the reason why a further significant increase in polyamines is not observed with HG treatment though there is a change in the activity of ODC. The source of polyamines in cells can be from uptake transporters, biosynthesis, or from intestinal flora.  In the case of cell cultures, it would be the former two. The media and sera used for the cells does contain amino acids which can contribute to intracellular levels. However, since glucose can modulate the levels of arginine and ornithine (as shown in Fig. 7), it indicates that the elevated cellular levels of polyamines (PUT) are contributed by the presence of glucose. While polyamine synthesis enzymes such as ODC, arginine decarboxylase, and agmatinase have been shown to be decreased in diabetic patients, and increased in breast cancer patients, there is not a measureable difference for patients with co-existing diabetes and breast cancer [8]. This is the first study to our knowledge to also report the metastatic potential of TNBC, MDA-MB-231 is significantly enhanced with HG treatments though DFMO is not protective towards this.
Glucose and polyamine production are related through the glycolytic/citric acid cycle pathway and the aspartateargininosuccinate shunt as shown in Fig. 7. This shunt is an important link deciding the fate of amino groups and carbon skeleton. It has been reported that glycolysis inhibition in neuroblastoma cells inhibits polyamine production and induces cell death, suggesting that glucose can modify polyamine levels through oxidative metabolism in cancer cells [36].
In conclusion, our data suggest that restricting polyamine (PUT) synthesis can be a plausible therapeutic option in preventing proliferation of breast cancer cells in Fig. 7 Link between glucose and polyamine through the aspartateargininosuccinate shunt. Glucose is metabolized through glycolysis (cytosol) and citric acid cycle (mitochondria). Oxaloacetate, a product of citric acid cycle can be shunted to form aspartate through transaminase which is further metabolized to argininosuccinate, a part of the urea cycle. Argininosuccinate is converted to arginine and then ornithine in the urea cycle, which are upstream substrates for conversion to polyamines diabetic states, while also affecting the growth profile of non-tumorigenic mammary epithelial cells (Fig. 8). Future studies would involve treating MDA-MB-231 cells for prolonged periods with HG to assess tumor formation in vitro using soft gel assays and in vivo using xenograft mouse models. In addition, effect of glucose on the progression in stages of breast cancer will be assessed using the same background MCF10A cell lines, i.e. normal MCF-10A, pre-malignant MCF-10AT, and malignant MCF-10CA1a. It is suggested that polyamine inhibitors can be combined with common breast cancer therapeutics in diabetic breast cancer patients, which can dramatically improve the prognosis of these patients. A comprehensive comparison of enzymes and metabolites of polyamine pathway in cancer patients with pre-existing diabetes in relation to those with cancer or diabetes alone, will help in customizing effective therapeutic regimen.