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Published in final edited form as: Gynecol Oncol. 2023 Dec 5;180:44–54. doi: 10.1016/j.ygyno.2023.11.013

Manipulation of metabolic responses enhances SHetA2 efficacy without toxicity in cervical cancer cell lines and xenografts

Rajani Rai a, Stanley Lightfoot b, Doris Mangiaracina Benbrook a,*
PMCID: PMC10922646  NIHMSID: NIHMS1949548  PMID: 38052108

Abstract

Objective:

The high frequency of cervical cancer recurrence after primary therapy necessitates alternative treatments. High-risk human papillomavirus (HR-HPV) causes cervical cancer and it’s continued presence supports elevated metabolism, proliferation and survival of cancer cells. The low-to-no toxicity new investigational drug, SHetA2, counteracts high-risk human papillomavirus (HR-HPV) effects on cell proliferation and survival in cervical cancer cells and xenograft tumors by disrupting heat shock protein 70 chaperone protection of oncogenic proteins. Our objective was to study the involvement of metabolism in SHetA2 effects on cervical cancer cells and tumors.

Methods:

SHetA2 proteomic and metabolic effects were measured in HR-HPV-positive CaSKi and SiHa and HR-HPV-negative C-33A cervical cancer cell lines. Combined treatment with 2-deoxyglucose (2-DG) was evaluated in cell culture and SiHa xenografts.

Results:

SHetA2 inhibited oxidative phosphorylation (OxPhos) and altered levels of proteins involved in metabolism, protein synthesis, and DNA replication and repair. Cervical cancer cells responded by elevating glycolysis. Inhibition of the glycolytic responses using galactose media or 2-DG increased SHetA2 sensitivity of two HR-HPV-positive, but not an HR-HPV-negative cervical cancer cell line. Interaction of 2-DG and SHetA2 was synergistic in HR-HPV positive cell lines in association with augmentation of SHetA2 ATP reduction, but not SHetA2 DNA damage induction. These results were verified in a SiHa xenograft tumor model without evidence of toxicity.

Conclusions:

Compensatory glycolysis counteracts OxPhos inhibition in SHetA2-treated HR-HPV-positive cervical cancer cell lines. Prevention of compensatory glycolysis with 2-DG or another glycolysis inhibitor has the potential to improve SHetA2 therapy without toxicity.

Keywords: Cervical cancer, oxidative phosphorylation, compensatory glycolysis, SHetA2, 2-deoxyglucose

1. Introduction

Approximately one in four cervical cancer patients suffers from recurrence following primary treatment [1]. Even patients diagnosed with early-stage cervical cancer experience cancer recurrence within 2–3 years after surgery at a rate of about 9 out of 100 [2]. In the United States, the median overall survival for recurrent and metastatic cervical cancer is approximately 16.5 months from the start of first-line therapy despite compliance with national treatment guidelines [3]. Furthermore, healthcare disparities worsen patient outcomes based on racial, ethnic, and regional factors [4, 5]. Disparities in low-middle-income countries result in cervical cancer being among the top killers of female populations [6].

The standard of care for cervical cancer consists of a combination of surgery, chemotherapy and radiation depending on disease severity. Patients presenting with advanced disease have the option of adding bevacizumab targeted at vascular endothelial growth factor or pembrolizumab targeted at programmed death-ligand 1 to their first-line treatment regimens, however, there remains a need to improve patient prognosis [3]. Given the high toxicities and morbidities caused by current therapies, targeting the elevated metabolism inherent in cancer is a current direction in new anti-cancer drug discovery and development strategies [7–10]. This is particularly relevant for cervical cancer because the vast majority of these tumors are caused by infection with high-risk human papillomavirus (HR-HPV). HR-HPV early oncoproteins cause metabolic reprogramming in cancer cells by interacting with cellular pathways to promote aerobic glycolysis [11]. Alterations in glucose, lipid and amino acid metabolism increase cervical cancer cell proliferation and survival in hostile microenvironments as they arise, metastasize and respond to therapeutic treatments [12].

The novel investigational new drug SHetA2, currently in clinical trial for cervical and other cancers (clinicaltrial.gov: NCT04928508), has the potential to improve cervical cancer therapy by targeting HR-HPV disrupted pathways to inhibit and kill cervical cancer cells without causing toxicity [13, 14]. We recently reported that the SHetA2 mechanism of inducing cell death is different in cervical cancer cells in comparison to multiple non-HPV associated cancers studied, including ovarian, endometrial, kidney, lung and head and neck [13]. The main differences involve significant contributions of caspase-independent apoptosis and mitophagy to SHetA2 induced cervical cancer cell death. Metabolic effects of SHetA2 in cervical cancer may also be different due to the effects of HR-HPV in this cancer type. In endometrial cancer cells, SHetA2 inhibited both oxidative phosphorylation (OxPhos) and glycolysis, putting cells into a quiescent state [15], however SHetA2 metabolic effects in cervical cancer are unknown.

The molecular mechanisms of SHetA2 involve its binding to three 70 kD heat shock protein proteins (HSP70s) called mortalin, heat shock cognate 70 (hsc70) and glucose regulated protein (Grp78), and disruption of their folding and support of client proteins and protein complexes [15–17]. SHetA2 interference with mortalin is the likely mechanism by which it caused mitochondrial damage and inhibits OxPhos, because mortalin is primarily located in the mitochondria where it serves a vital role in nuclear-encoded protein import and mitochondrial function [18]. Other client proteins released from mortalin by SHetA2 treatment of endometrial cancer cells include the metabolic enzymes [15]. In cervical cancer cell lines, SHetA2 disruption of hsc70/apoptosis inducing factor (AIF) complexes is associated with nuclear accumulation of AIF and DNA damage [13].

In this study, we evaluated the proteomic and metabolic responses of cervical cancer cells to SHetA2, identified a metabolic-targeted strategy to increase SHetA2 sensitivity of these cell lines, and validated the results in vivo.

2. Methods

2.1. Cell culture and chemicals

HR-HPV-positive (SiHa, CaSKi) and HR-HPV-negative (C-33A) human cervical cancer cell lines were cultured as previously described [13, 14]. SHetA2 synthesized by K. Darrell Berlin, PhD, (Oklahoma State University, Stillwater, OK, USA) as described [19] was dissolved in dimethlyl sulfoxide (DMSO) for cell culture studies. SHetA2 synthesized by the US National Cancer Institute RAID Program was suspended in 30% Kolliphor HS 15 (SigmaAldrich, Merck, Darmstadt, Germany) for animal studies. 2-deoxy-D-glucose (2-DG #D8375, Sigma-Aldrich) was dissolved in water.

2.2. Mass spectrometry proteomic analysis

Cultures were treated with 10 μM SHetA2 or DMSO in triplicate for 24 h. Proteins were isolated with mPER (#78501, Thermofisher, Waltham, MA, USA) and evaluated by liquid chromatography-mass spectrometry as previously described [20].

2.3. Seahorse metabolism assays

OxPhos and glycolysis were measured using XF Cell Mito Stress test and XF Glycolytic Rate Assay, respectively (Agilent Bioscience, Santa Clara, CA, USA). Briefly, cells grown on XF96 tissue culture plates were treated with 10 μM SHetA2 for 4 h, washed with assay medium and incubated at 37°C for 45 min. For the Mito Stress test, oligomycin (1 μM), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (2 μM) and a mixture of antimycin A and rotenone (0.5 μM each) were added into appropriate cartridge ports and the instrument was calibrated. Then, the cell culture plate was loaded into the instrument and oxygen consumption rate (OCR) measured. For the glycolytic rate assay, 2-DG (50 mM), and a mixture of antimycin A and rotenone (0.5 μM each) were into appropriate cartridge ports. After cartridge calibration, the plate was washed with assay medium, loaded into the instrument and the Seahorse XF Glycolytic Rate Assay Report Generator was used to calculate extracellular acidification rate (ECAR) and proton efflux rate (PER). All data were normalized to total protein.

2.4. Metabolic Viability Assay

Metabolic viability was measured using an MTT assay (#G4100, Promega Madison, WI, USA). Briefly, solution was added. Optical density (OD) was measured using a BioTek Synergy H1 Micro Plate Reader. The average ODs of triplicate treatments were normalized to the average ODs of the respective controls. GraphPad Prism 10 software was used to derive the half maximal inhibitory concentrations (IC50s/potencies), and efficacies (maximal percent growth inhibition activities).

2.5. ATP Production

Monolayer cultures incubated at 10,000 cells/well in 96-well culture plates for 24 h, or spheroids generated by culturing 2000 cells/well in 96-well clear round bottom ultra-low attachment microplates (#7007, Corning) for 48 h were treated with various SHetA2 concentrations, alone or in combination with 2-DG (0.25mM) for 24 h. ATP production was measure in the monolayer or spheroid cultures with the CellTiter-Glo 2.0 Cell Viability or CellTiter-Glo 3D Cell Viability Assay (#G9241 or #G9683, respectively, Promega, Madison, WI, USA). Relative luminescence units were calculated by normalizing luminescence units of treated to controls treated with solvent only.

2.6. Drug Interactions

Cells cultured in 96-well plates were treated in triplicate with a two-fold dilution series of a 1:1 ratio of SHetA2 to 2-DG IC50 values for 72 hrs. Fold effects for each treatment were determined using the MTT assay and calculated by dividing average ODs of treated cultures by the average OD of control cultures treated with solvent only and then subtracting this quotient from number 1. The average fold effects of each dose were used to draw isobolograms and calculate combination index (CI) values at three effective doses (ED’s) that caused 50%, 75%, 90%, or 95% cell reductions (ED50, ED75, ED90 or ED95, respectively) using CompuSyn Software based on the Chou-Talalay Method [21].

2.7. Fluorescent Immunocytochemical Staining and Imaging

Approximately 6–8000 cells plated onto 8-chambered slides were treated with SHetA2, 2-DG or both for 48 h, followed by fixation with 4% paraformaldehyde, permeabilization with 0.1% TritonX 100, blocking with 4% bovine serum albumin, and sequential incubation with MitoTracker™ Red CMXRos (#M7512, Thermo Fisher Scientific, Waltham, MS, USA) for 45 min, primary antibody for γH2AX (#2577, Cell signaling, 1:100 dilutions) overnight, Alexa Fluor 488 labeled secondary antibody for 1 h and DAPI. Images were acquired with a 63X objective using a Zeiss Axio Observer Z1 (Göttingen, Germany).

2.8. Xenograft Tumor Drug Interaction Study

This animal study was approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee (IACUC Protocol #19–009-CHI). Four-week-old female athymic mice (Hsd:Athymic Nude-Foxn1nu, ENVIGO, Indianapolis, IN, USA) were subcutaneously injected with 1.2 × 107 SiHa cells. Tumor sizes were measured thrice/week. Body weights were measured weekly. Once tumors achieved ~50 mm3 volume ([width2 × length]/2), mice were randomized based on tumor sizes into four groups (N=8). Placebo or SHetA2 60 mg/kg was administered daily for 31 days by oral gavage. 2-DG was given in drinking water. At treatment termination, animals were euthanized, and tumors and organs were collected and weighed.

2.9. Evaluation of Organs and Tumors

Organ and tumor tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemistry for phosphorylated histone γH2AX (1:50 dilution, # 9718, Cell Signaling Technology, Danvers, MA) as previously described [14]. ImageJ software was used to count the number of positively-stained nuclei in digital micrographs taken at the same magnification of a tumor-filled area with strong positive staining for each of six tumors in each treatment group. Hematoxylin and eosin-stained sections of the organs were evaluated by an experienced pathologist (S.L.).

2.10. Statistical analysis

All experiments were independently repeated at least twice in triplicate. Data are expressed as mean ± standard deviation (SD) for experimental replicates or ± standard deviation of the mean (SEM) for biological replicates. T-tests or ANOVAs were used to compare two groups or multiple groups, respectively. p <0.05 was considered statistically significant. Multiple paired t-test analysis was used to identify proteins with q values <0.05 (<5% false discovery rate) in triplicate results of protein levels in treated compared to control cultures. Statistical analyses were performed using GraphPad Prism 9 Software (GraphPad Software Inc., La Jolla, CA, USA).

3. Results

3.1. SHetA2 alters proteins involved in metabolism, protein synthesis, and DNA replication and repair

SHetA2 does not affect cellular levels of its direct binding proteins, but instead releases client proteins from the SHetA2-binding proteins, often resulting in ubiquitin-mediated degradation and/or cellular re-localization of the client proteins [17, 22]. In this study, SHetA2-induced alterations in protein levels were profiled by mass spectrometry analysis of C-33A and SiHa cultures treated for 24 h with 10 μM SHetA2 versus vehicle. C-33A cells responded by upregulating 15 and reducing 3 proteins (Supplemental Table 1). Six of these 18 (33%) proteins are involved in mRNA translation (ribosomal proteins L4, 7A, L10L and S2; valyl-tRNA synthetase 1/VARS1; and poly(U) binding splicing factor 60/PUF60 [23]. SHetA2 reduced glutamic-oxaloacetic transaminase 2 (GOT2), which catalyzes the reversible interconversion of oxaloacetate and glutamate to aspartate and α-ketoglutarate in the malate-aspartate shuttle, and increased pyruvate carboxylase, which processes the pyruvate end product of glycolysis into oxaloacetate, which feeds into OxPhos. Only one of these 18 proteins, is involved in DNA repair: hepatoma-derived growth factor, which promotes homologous recombination repair of DNA double stranded breaks [24].

SiHa cells responded to SHetA2 by reducing the expression of eight and upregulating that of three proteins (Supplemental Table 1). Five of these 11 (46%) proteins are involved in mRNA translation (ribosomal proteins L10a, L36, S23, S4 X-linked, and FtsJ RNA 2’-O-methyltransferase 3/FtsJ3, involved in ribosome biogenesis [25]. Four of the 11 (28%) proteins (basic transcription factor 3/BTF3, DNA replication helicase/nuclease 2, protein phosphatase 2 phosphatase activator, and synuclein alpha) are directly or indirectly involved in DNA damage repair. There were no proteins significantly modulated by SHetA2 in common between the two cell lines.

Ingenuity Pathway Analysis (IPA, Qiagen Digital Insights) demonstrated that the majority of the SHetA2-altered proteins in each cell line are directly bound by the SHetA2 target proteins Grp78, hsc70 and mortalin (Fig. 1). These SHetA2-binding and SHetA2-modulated proteins in cervical cancer cells connect through an integrated network predominantly involved in pathways needed to support heightened protein production and DNA replication in both cell lines and DNA damage repair in SiHa.

Figure 1. Network connections of SHetA2-regulated proteins with SHetA2-binding proteins.

Figure 1.

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IPA analysis of proteins significantly regulated by SHetA2 in C33-A (upper panel) and SiHa (lower panel) cervical cancer cell lines. Red indicates upregulation of protein by SHetA2 treatment. Green indicates down-regulation of protein by SHetA2 treatment. Blue indicates SHetA2-binding proteins.

3.2. Cervical cancer cells increase glycolysis in response to SHetA2 OxPhos inhibition

In all three cell lines, 4 hr treatment with 10 μM SHetA2 stopped OxPhos, as indicated by reduced OCR in Seahorse assays (Fig. 2A). SHetA2 reduced basal respiration (Fig. 2B), and completely blocked ATP production (Fig. 2C) and maximal respiration (Fig. 2D). SHetA2 also reduced spare respiratory capacity (SRC; extra mitochondrial capacity to produce energy under stress or nutrient-deprived conditions Fig. 2E), and proton leak (an indicator of mitochondrial damage or a cellular response that regulates mitochondrial ATP production Fig. F).

Figure 2. SHetA2 inhibits OxPhos in cervical cancer cell lines:

Figure 2.

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A) Cervical cancer cells were treated with 10 μM SHetA2 for 4 hour and OCR was measured using the Seahorse XFe96 analyzer and cell mito-stress assay. B) Basal respiration (OCR, measured at starting point before injection of any substance representing the basic cellular ATP demand), C) ATP production (OCR measured after the injection of the ATP synthase inhibitor oligomycin representing ATP produced by the mitochondria that contributes to meeting the energetic needs of the cell), D) Maximal Respiration (OCR measured after adding FCCP/uncoupler), E) Spare respiratory capacity and F) Proton leak (Remaining basal OCR not coupled to ATP production after addition of antimycin A and rotenone) are shown. Data are shown as mean±SD. **** p ≤ 0.0001 when compared with respective control.

In contrast, this same SHetA2 treatment regime caused increased ECAR in all three cell lines (Fig. 3A). SHetA2 significantly increased basal PER (Fig. 3B), which includes both PER due to OxPhos and glycolysis; however, the effect of SHetA2 was mainly due to increased contribution of glycolysis as indicated by GlycoPER (Fig. 3C). SHetA2 induction of glycolysis achieved equivalent or greater levels than the extra compensatory glycolysis induced by combination of rotenone and antimycin A treatment seen at 20 min on the x-axis. The level of SHetA2 induction of extra compensatory glycolysis above that induced by rotenone and antimycin A was statistically significant in the HR-HPV negative, but not in the HR-HPV positive, cell lines (Fig. 3D). An energetic map of these data illustrates that SHetA2 decreased OxPhos and increased glycolysis in all three cell lines (Fig. 3E) with a maximum metabolic switch observed in SiHa. SHetA2 significantly reduced the ratios of OxPhos OCR/glycolysis ECAR (Fig. 3F) and ATP produced by OxPhos to that produced by glycolysis (Fig. 3G) in all cell lines.

Figure 3. Cervical cancer cells respond to SHetA2 by increasing the glycolysis.

Figure 3.

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A) Cervical cancer cells were treated with SHetA2 for 4 hours and ECAR was measured using the Seahorse XF-96 analyzer and glycolytic rate assay. B) Basal PER, representing (total protons exported by cells measured representing the basic cellular ATP demand), C) Basal glycol PER (PER derived from glycolysis), and D) extra compensatory glycolysis (The rate of glycolysis in cells following the addition of Rotenone and Antimycin A to effectively inhibit oxidative phosphorylation and drive compensatory changes in the cell to use glycolysis to meet the cells’ energy demands) were estimated using the XF Glycolytic Rate Assay Report Generator. Data are shown as mean±SD and analyzed with a t-test. (E) The energetic map of cervical cancer cells was assessed by XF Glycolytic Rate Assay Report. Ratios of OCR/ ECAR (F) and mito/glyco ATP (G) were calculated and shown. ** p ≤ 0.01, **** p ≤ 0.0001 when compared with respective controls.

3.3. Prevention of the glycolytic response increases SHetA2 sensitivity

To evaluate the role of compensatory glycolysis in SHetA2 resistance, we used a medium that cannot fuel glycolysis (Fig. 4A). Cells grown in glucose-medium can utilize both OxPhos and glycolysis to produce ATP, whereas cells grown in galactose-medium predominantly produce ATP through OxPhos. SHetA2 sensitivity of the HR-HPV negative C-33A cell line was similar in glucose and galactose media (IC50’s ~3 μM). Potencies of SHetA2 in HR-HPV positive CaSKi and SiHa cell lines were lower (higher IC50’s) in galactose compared to glucose media. SHetA2 efficacy was higher in SiHa in galactose medium compared to glucose media.

Figure 4. Inhibition of the glycolytic response increases SHetA2 sensitivity:

Figure 4.

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A) Representative dose-response curves and IC50 values of cervical cancer cells treated with SHetA2 (0–10 μM) for 72 hours in galactose vs. glucose media and assessed for metabolic viability using an MTT assay. B) Representative dose-response curves and IC50 values of cervical cancer cells treated with various doses of SHetA2 or combination of SHetA2 and 0.25 mM of 2-deoxyglucose (2-DG) for 72 hours. C-E) Isobologram of SHetA2 and 2-DG combinations (Dose A: SHetA2, Dose B: 2-DG, C), ATP assay (D) and spheroid viability assay (E) in CaSKi cervical cancer cells treated with different concentrations of the SHetA2 alone or in combination with 2-DG. F-H) Isobologram of SHetA2 and 2-DG combinations in SiHa cervical cancer cells (Dose A: SHetA2, Dose B: 2-DG, F), ATP assay (G) Spheroid viability assay (H) and on SiHa cells treated with different concentrations of the SHetA2 alone or in combination with 2-DG.

To translate these results toward clinical trial, effects of inhibiting glycolysis with physiologically-achievable concentrations of 2-DG [26] on SHetA2 sensitivities of the cell lines were measured. Similar to the results in galactose media, 2-DG altered SHetA2 potencies in HR-HPV positive CaSKi and SiHa, but not in HR-HPV negative C-33A cells (Fig. 4B). CI values derived for ED50, ED75 and ED90 were below the additive line with a value of 1, indicating synergy of SHetA2 and 2-DG in both CaSKi and SiHa (Fig. 4C, F). For both cell lines, all CI values indicated synergy, while strong synergy was indicated for SiHa at ED95. The DRI’s demonstrate that when the when drugs are used in combination, a reduced amount of the drugs could be used to achieve the same efficacy (Supplemental Table 2).

3.4. Involvement of ATP production and DNA damage in the synergy mechanism

SHetA2 inhibition of ATP production was augmented by co-treatment with 2-DG over a range of doses in SiHa and CaSKi cell lines when grown in 2-D cultures (Fig. 4D,G) and as 3D spheroids (Fig. 4E,F). SHetA2-induction of nuclear γH2AX staining as an indicator of DNA double-stranded breaks was not affected by 2-DG in either cell line (Fig.5).

Figure 5. SHetA2-induced DNA damage is not prevented by 2-DG:

Figure 5.

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CaSKi and SiHa cells were treated with either SHetA2, 2-DG or their combination for 24 hours and stained with MitoTracker™ Red CMXRos, γH2AX (green) and DAPI (blue) followed by confocal microscopy to check protein expression. Representative immunofluorescence images were shown (upper panel). Image J software was used to quantify nuclear expression of γH2AX and shown as bar graph (lower panel).

3.5. In vivo Validation of Drug Combination Efficacy and Mechanism

To validate and translate our in vitro findings, we demonstrated that the SHetA2 and 2-DG drug combination reduced volumes (Fig. 6A) and final weights (Fig. 6B) of SiHa xenograft tumors in comparison to single drug or untreated control groups, without significantly altering body weights (Fig. 6C). There was no evidence of drug toxicity in the liver, kidney or spleen histology (Fig. 6D) or weights (Supplemental Table 3). Quantification of nuclear γH2AX staining of tumors confirmed that 2-DG did not significantly increase SHetA2-induced DNA damage (Fig. 6E).

Figure 6. 2-DG enhances SHetA2 efficacy in SiHa xenograft model without toxicity:

Figure 6.

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Effect of SHetA2, 2-DG and combination treatment on average tumor volume (A), tumor weight (B) and body weight (C) in SiHa xenograft tumor bearing mice. D) Representative images of- H&E staining on liver, kidney and spleen tissue, and -γH2AX IHC staining on tumor tissue collected from mice in different treatment group. E) Quantification of γH2AX staining by image J software. * p ≤ 0.05, ** p ≤ 0.01 when compared with respective control.

4.0. Discussion

Although cervical cancer is preventable, it is far from eradicated, due to low vaccination rates against the HR-HPV causative factor, cancer recurrence, and drug resistance. Previously, we demonstrated that the novel investigational new drug SHetA2 inhibits HR-HPV-regulated pathways and causes a distinct mechanism of cell death in cervical cancer that is caspase-independent, whereas the mechanisms in other cancer types involve caspases [13, 27, 28]. It is plausible that HR-HPV in cervical cancer is responsible for this distinguishing mechanism of cell death, and could also drive distinguishing metabolic responses to various therapies [29]. Here, we demonstrated that cervical cancer cell lines exhibit reduced OxPhos and increased glycolysis in response to treatment with SHetA2. This cervical cancer response is different from the reduction in both OxPhos and glycolysis observed in endometrial cancer cell lines [15].

The direct molecular mechanism of SHetA2 involves binding to Grp78, hsc70 and mortalin chaperones leading to disruption of their complexes with, and protection of, client proteins [13, 15–17]. In this study, the majority of proteins identified to be present at significantly different levels in SHetA2-treated cells compared to control cells are known to be bound by these SHetA2 targeted chaperones and involved in regulation of protein synthesis and DNA damage repair. Reduction of OxPhos is a logical consequence of SHetA2 interference with mortalin and mitochondrial function leading to mitophagy and relocation of AIF from the mitochondria to the nucleus where it promotes DNA damage. However, interference with mitophagy and/or AIF expression only partially reduced SHetA2-induced cervical cancer cell death [13], indicating the contribution of other factors to the mechanism of cell death.

In this study, we demonstrate that elevated glycolysis can compensate for SHetA2’s inhibition of ATP production and tumor growth. Although the upregulation of glycolysis can compensate for reduced OxPhos ATP production, it does so at a lower efficiency. In this study, we demonstrated that SHetA2-induced glycolysis serves a compensatory function in two HR-HPV positive cervical cancer cell lines, CaSKi and SiHa, but not in the HR-HPV negative cell line C-33A. Because C-33A has the highest sensitivity to SHetA2 compared to SiHa and CaSKi [13], the ability of SHetA2 to kill the cell line may already be maximized.

There are also several molecular mechanisms that could explain why C-33A is not sensitized to SHetA2 by glycolysis, including its HR-HPV and p53 status and proteomic response to SHetA2 treatment. A metabolomic study found that HR-HPV positive SiHa and HeLa cell lines were more glycolytic than HR-HPV negative C-33A cancerous and HCKIT non-cancerous cell lines, and that C-33A had elevated arginine metabolism [30]. In addition, the R273C p53 mutation in C-33A may contribute to the lack of sensitization through its rewiring of metabolism. The similar R273H p53 mutation and other hot-spot p53 missense mutations have been shown to decrease OxPhos and increase glycolysis in breast cancer cells [31]. In the current study, the inability of glycolysis inhibition to sensitize the C-33A cell line to SHetA2, may also be related to the SHetA2 inhibition of GOT2 protein levels observed in C-33A cells. GOT2 is the intra-mitochondrial form of GOT that is important for bringing NADH produced by glycolysis into the mitochondria for regeneration into NAD+ [32]. The SHetA2 reduction of GOT2 in C-33A may be caused by SHetA2 releasing GOT2 from hsc70 protection from degradation. GOT2 folding and import into the mitochondria are regulated by a protein recognized by the N27F3–4 antibody that binds to both HSP70 and hsc70 [33]. Previously, we demonstrated that SHetA2 can disrupt hsc70/AIF complexes and identified hsc70 as a relevant target for the treatment of cervical cancer via association of its overexpression with cervical cancer patient survival probability and inhibition of AIF [13]. All of these factors may contribute to C-33A’s greater sensitivity to SHetA2 and reduced reliance on glycolysis compared to the HR-HPV positive cervical cancer cell lines CaSKi and SiHa, thereby over-riding the ability of glycolysis inhibition to sensitize C-33A to SHetA2.

In this study, the ability of glycolysis to compensate for SHetA2 inhibition of ATP production was demonstrated in HR-HPV positive cervical cancer cell lines using two methods. Both use of galactose media or 2-DG to prevent glycolysis enhanced SHetA2 reduction of mitochondrial metabolic viability and ATP production. The enhanced inhibition of mitochondrial metabolic viability is likely due to 2-DG reduction of the glycolysis product pyruvate feeding into mitochondrial metabolism. The mechanism of 2-DG enhancement of SHetA2 did not involve DNA damage.

The ongoing SHetA2 Phase 1 clinical trial (clinicaltrials.gov: NCT04928508) and extensive clinical use of 2-DG offer a pathway for rapid translation to SHetA2-based drug combination clinical trials. A Phase 1 trial of oral 2-DG in combination with docetaxel chemotherapy in patients with advanced solid tumors identified a safe starting dose [26]. As SHetA2 is also orally bioavailable, patients treated with the SHetA2 and 2-DG combination could take their drugs at home. Concerns for potential toxicity caused by SHetA2 damage to mitochondria are reduced by the established SHetA2-resistance of mitochondria in non-cancer cells [34] and the lack of 12-week SHetA2 treatment effects on heart function [35]. Overall SHetA2 caused no skin irritancy, mutagenicity, carcinogenicity or any observable toxicity at doses 50-fold higher than the predicted therapeutic dose [36–40]. Thus, the combination of SHetA2 with 2-DG represents a rational combination therapy that could be more effective than SHetA2 treatment alone and without added toxicity.

A limitation of this study is that it evaluated only one HR-HPV negative cell line. HR-HPV independent cervical tumors are rare and the availability of HPV-negative cervical cancer cell lines is limited. Additional cell lines, primary cultures and patient derived xenografts from HR-HPV independent tumors is needed to conclude that the differences in C-33A compared to CaSKi and SiHa observed in this study are due to the lack of HR-HPV in C-33A.

5.0. Conclusion

Our study demonstrates that elevated glycolysis partially compensates for reduced OxPhos in SHetA2-treated HR-HPV positive cervical cancer cells. The compensatory glycolysis can be counteracted with culture media that does not support glycolysis or with 2-DG co-treatment in vitro. These results were validated in an in vivo xenograft model, supporting further study and translation of the combination treatment of SHetA2 and other OxPhos inhibiting cancer therapeutics with 2-DG and other glycolysis inhibitors.

Supplementary Material

1

Supplemental Table 1. Proteins Altered in C33A and SiHa Cervical Cancer Cell Lines by SHetA2 with >5% False Discovery Rates.

2

Supplemental Table 2. DRI and CI values for the SHetA2 and 2-DG Combination in Cervical Cancer Cell Lines.

3

Supplemental Table 3. Organ to Body Weight Ratios of Animals in Different Treatment Groups.

Highlights.

  • SHetA2 inhibits oxidative phosphorylation in cervical cancer cells and inhibits tumor growth

  • Cervical cancer cells respond by increasing glycolysis

  • Inhibition of glycolysis increases SHetA2 reduction of cervical cancer cell metabolic viability

  • 2-deoxyglucose synergizes with SHetA2 reduction of metabolic viability in cervical cancer cells

  • 2-deoxyglucose significantly enhances SHetA2 tumor growth inhibition without toxicity to mice

Acknowledgements

Research reported in this publication utilized Institutional Research Core facility for Molecular Biology and Cytometry and Tissue Pathology and Biospecimen Shared Resources of the Stephenson Cancer Center. We thank Dr. Vishal Chandra for fluorescent cell imaging and analysis, and Swati Chaudhari and Dr. Debasish Dey for their assistance with the animal model. Illustration created with BioRender.com. We thank Virginie Sjoelund for advice about mass spectrometry experiments and uploading the data to a public data repository.

Research reported in this publication was supported by National Cancer Institute (NCI) grant R01CA200126 (D.M.B) and in part by the NCI Cancer Center Support Grant P30CA225520 awarded to the University of Oklahoma Stephenson Cancer Center and used the Molecular Biology and Cytometry Research and the Biospecimen and Tissue Pathology Shared Resources.

Abbreviations

2-DG

2-deoxyglucose

AIF

apoptosis-inducing factor

ANOVA

analysis of variance

CI

combination index

DMSO

dimethylsulfoxide

DRI

dose reduction index

ECAR

extracellular acidification rate

ED

effective dose

FCCP

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

FFPE

formalin-fixed and paraffin-embedded

GlycoPER

proton efflux rate caused by glycolysis

GOT2

glutamic-oxaloacetic transaminase 2

Grp78

glucose regulated protein 78

HR-HPV

high risk human papillomavirus

hsc70

heat shock cognate 70, IACUC, Institutional Animal Care and Use Committee

IC50

half maximal inhibitory concentration

MTT

3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay

OCR

oxygen consumption rate

OD

optical density

OxPhos

oxidative phosphorylation

PER

proton efflux rate

SCR

spare respiratory capacity

SD

standard deviation

SEM

standard error of the mean

Footnotes

The authors declare no potential conflicts of interest.

Declaration of interests

The authors declare no competing interests.

Ethics approval

The animal model was approved by the University of Oklahoma Health Sciences Center IACUC.

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Availability of data and materials

Proteomic data is available on ProteomeXchange: http://www.ebi.ac.uk/pride/archive/projects/PXD029537.

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Associated Data

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

Supplementary Materials

1

Supplemental Table 1. Proteins Altered in C33A and SiHa Cervical Cancer Cell Lines by SHetA2 with >5% False Discovery Rates.

NIHMS1949548-supplement-1.xls (36KB, xls)
2

Supplemental Table 2. DRI and CI values for the SHetA2 and 2-DG Combination in Cervical Cancer Cell Lines.

NIHMS1949548-supplement-2.docx (14.7KB, docx)
3

Supplemental Table 3. Organ to Body Weight Ratios of Animals in Different Treatment Groups.

NIHMS1949548-supplement-3.docx (14.4KB, docx)

Data Availability Statement

Proteomic data is available on ProteomeXchange: http://www.ebi.ac.uk/pride/archive/projects/PXD029537.

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