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Immunohistochemical Analysis of the Metabolic Phenotype of Adrenal Cortical Carcinoma
Kai Duan 1,2 . Hasan Gucer3 . Mehmet Kefeli4 · Sylvia L. Asa 1,5 . Daniel A. Winer 1,2,6,7 . Ozgur Mete 1,2
C Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Metabolic reprogramming is a cellular process contributing to carcinogenesis. However, it remains poorly understood in adrenal cortical carcinoma (ACC), an aggressive malignancy with overall poor prognosis and limited therapeutic op- tions. We characterized the metabolic phenotype of ACC, by examining the immunoprofile of key proteins involved in glucose metabolism, hexokinase (HK1), pyruvate kinase (PKM1, PKM2), succinate dehydrogenase (SDHB), and phospho-S6 ribosomal protein (pS6), in a tissue microarray of 137 adrenal cortical tissue samples. Protein expression was compared between ACC (n =42), adrenal cortical adenoma (ACA; n =50), and normal adrenal cortical tissue samples (n =45). Cytoplasmic expression of HK1 and PKM2 was significantly higher in ACC than in ACA (p < 0.001 and p =0.014, respectively) or normal adrenal cortical tissue samples (p <0.001 and p <0.001, respectively). Expression of HK1 and PKM2 was also higher in ACA than in normal adrenal cortical tissue samples (p <0.001 and p < 0.001, respectively). PKM1 expression was overall low in ACC, ACA, and normal samples, although expression of PKM1 was higher in ACC than in ACA (p =0.027). There was no loss of cytoplasmic granular SDHB expression in our cohort of adrenal cortical tumors, and cytoplasmic expression of pS6 was lower in ACC than in ACA (p =0.003) or normal adrenal cortical tissue samples (p =0.008). Significantly, HK1 expression correlated with pyruvate kinase isoform (PKM2 and PKM1) expression (p <0.001 and p =0.007, respectively). Although functional validation was not performed, this study provides further evidence that metabolic reprogramming and altered glucose metabolism may occur in a subset of ACC through overexpression of intracellular glycolytic enzymes, notably HK1 and PKM2. The possibility of utilizing the reprogrammed glucose metabolism in ACC for novel therapeutic strategies should be explored in future studies.
Keywords Adrenal cortical carcinoma · Adrenal cortical adenoma · Metabolic profiling · Altered glucose metabolism · Glycolysis
☒ Ozgur Mete
1 Department of Pathology, University Health Network, 200 Elizabeth Street, 11th floor, Toronto, ON M5G 2C4, Canada
2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
3 Department of Pathology, Recep Tayyip Erdogan University, Rize, Turkey
4 Department of Pathology, Ondokuz Mayis University, Samsun, Turkey
5 Department of Pathology, University Hospitals Cleveland Medical Center and Case Western Reserve University, Cleveland, OH, USA
6 Division of Cellular & Molecular Biology, Diabetes Research Group, Toronto General Hospital Research Institute (TGHRI), University Health Network, Toronto, Canada
7 Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA, USA
Introduction
Adrenal cortical carcinoma (ACC) is a rare endocrine malig- nancy with frequent aggressive biology and overall poor prog- nosis [1-3]. Although our understanding of this disease has progressed tremendously over the last decade, our knowledge of ACC pathogenesis remains incomplete and new therapeutic avenues are needed [1-7].
Recently, several groups have begun to scrutinize the role of metabolic reprogramming in the pathogenesis of ACC [8-10]. Altered glucose metabolism has been de- scribed in many cancers and is thought to be a necessary step to fulfill the anabolic demands of proliferating tumor cells [11-14]. First described over 80 years ago, aerobic glycolysis (also known as the “Warburg” effect) refers to a propensity for proliferating tumor cells to acquire a hyperglycolytic phenotype, whereby glucose is taken up
a
Glucose
1
GLUT
Glucose
HK1
Glucose-6-phosphate
Phosphoenolpyruvate
PKM1, PKM2
SDHB
Pyruvate
TCA cycle
Lactate 1
MCT
*Simplified Figure of Glucose Metabolism in Normal Cell
b
Glucose 1
GLUT
Glucose
HK1
PI3k-AKT-mTOR
Glucose-6-phosphate
p53
Phosphoenolpyruvate
PKM1, PKM2
SDHB
Pyruvate
TCA cycle
Lactate
MCT
*Simplified Figure of Glucose Metabolism in Cancer Cell
and preferentially converted into lactate, even in the pres- ence of oxygen and functioning mitochondria [11-16] (Fig. 1). Although this adaptive phenomenon has been observed in many cancers, it remains poorly understood in ACC [8-11, 17, 18]. A deeper understanding of ACC metabolism may allow for more effective targeted thera- peutic strategies to benefit patients in the future [12, 14, 19].
In order to characterize the metabolic phenotype of ACC, we investigated the immunoprofile of key proteins involved in glucose energy metabolism. We developed an immunohistochemical panel that would allow for screen- ing of key markers to provide a snapshot of metabolism in the stained tissues. These proteins were designed to cover
the glycolytic enzyme hexokinase I (HK1), and pyruvate kinase M1 and M2 isoforms (PKM1 and PKM2), as well as mitochondrial proteins involved in the citric acid cycle using staining for succinate dehydrogenase (SDHB); while SDHB expression is well known to reflect the ge- netic integrity of the various members of the SDH com- plex, in this study, we used it as a biomarker that can reflect SDH-related pseudohypoxia. The panel also in- cluded phospho-S6 ribosomal protein (pS6), a marker of growth response to anabolic orchestrators like mammalian target of rapamycin (mTOR). These markers were assessed in a surgical series of benign and malignant ad- renal cortical neoplasms, as well as normal adrenal corti- cal tissue.
Material and Methods
Study Group and Histology
We evaluated a series of tissue microarrays (TMA), construct- ed at the University Health Network (UHN), as reported in a previous study [7]. Briefly, after obtaining the approval of the institutional Research Ethics Board, a total of 137 surgical specimens (42 adrenal cortical carcinomas (ACC), 50 adrenal cortical adenomas (ACA), and 45 normal adrenal cortical tis- sue samples) were selected for the construction of TMA. All cases were retrospectively reviewed by 3 endocrine patholo- gists (O.M., H.G., and S.L.A.) to ensure the appropriate inclu- sion of the diagnostic categories.
Tissue Microarray Construction
Reference hematoxylin and eosin-stained slides for each pa- tient were reviewed and marked with at least 3 circles (each measuring 1 mm in diameter) to select regions without necro- sis. Three cores of 1 mm diameter were taken from the corre- sponding paraffin block matching the circled regions. The cores were then mounted in linear arrays in recipient paraffin blocks using a manual tissue arrayer. At least 80% viable cells were obtained for each sample. A character map was con- structed delineating the various cases and their order in the TMA.
Immunohistochemistry
Formalin-fixed paraffin-embedded sections (4 um) were dewaxed in 5 changes of xylene and rehydrated through grad- ed alcohols. The details of the antibodies used are summarized in Table 1. Negative and positive control tissues were selected based on manufacturer recommendations as well as previous publications where these antibodies were applied. Multiple control experiments were undertaken to optimize each anti- body. Endogenous peroxidase was blocked with 3% hydrogen peroxide. The detection system used was MACH4 universal HRP polymer system (Inter Medico; cat BC-M4U534). Color development was performed with freshly prepared DAB (DAKO; cat K3468). Sections were counterstained
lightly with Mayer’s hematoxylin, dehydrated in alcohols, cleared in xylene, and mounted with Permount mounting me- dium (Fisher; cat SP15-500). Individual cores were scored by multiplying the percent positive cells by intensity scores (1 to 3) and each case received an average score (max 300) and an overall staining intensity (max 3) for HK1, PKM1, PKM2, and pS6. Levels of cytoplasmic granular staining of SDHB expression were recorded.
Statistical Analysis
The metabolic biomarker (HK1, PKM2, PKM1, pS6) expres- sion scores and SDHB expression were compared between ACCs, ACAs, and normal adrenal cortical tissue samples. The available data on mitotic tumor grade and Ki67 labeling indices [7] were also correlated with metabolic biomarkers in ACCs. Statistical analyses were performed using the SPSS statistical software program (SPSS for Windows release 19.0; SPSS Inc., Chicago, IL).
Results
Expression Profile of Proteins Involved in Glycolysis
The expression scores are summarized in Table 2. Overall, HK1 and PKM2 expression differed significantly between ACC (n =42), ACA (n =50), and normal adrenal cortical tissue samples (n =45) (p <0.001 and p <0.001, respective- ly). Intermediate-to-high intensity cytoplasmic HK1 expres- sion was seen in 62% of ACC versus 20% of ACA versus 13% of normal adrenal cortical tissue samples (p <0.001). The mean HK1 expression scores were 146.20 in ACC versus 58.62 in ACAs versus 22.52 in normal adrenal cortical tissue samples. Cytoplasmic expression of HK1 was significantly higher in ACC than in ACA (p <0.001) or normal adrenal cortical tissue samples (p <0.001) (Fig. 2). Expression of HK1 was also higher in ACA than in normal adrenal cortical tissue samples (p <0.001). Similarly, intermediate-to-high in- tensity cytoplasmic PKM2 expression was seen in 79% of ACC versus 60% of ACA versus 47% of normal adrenal cor- tical tissue samples (p =0.001). The mean PKM2 expression
| Antibody | Clone | Source | Dilution |
|---|---|---|---|
| Hexokinase I (HK1) | C35C4 | Cell Signaling Technology | 1:400 |
| Pyruvate kinase M2 isoform (PKM2) | D78A4 | Cell Signaling Technology | 1:1500 |
| Pyruvate kinase M1 isoform (PKM1) | D30G6 | Cell Signaling Technology | 1:200 |
| Phospho-S6 ribosomal protein (pS6) | Ser235/236 | Cell Signaling Technology | 1:100 |
| Succinate dehydrogenase B (SDHB) | Polyclonal | Sigma-Aldrich | 1:700 |
| Biomarkers | ACC | ACA | Normal tissue | p |
|---|---|---|---|---|
| HK1 | ||||
| Mean expression score (max 300) | 146.20 | 58.62 | 22.52 | <0.001 |
| PKM2 | ||||
| Mean expression score (max 300) | 203.24 | 156.56 | 100.20 | <0.001 |
| PKM1 | ||||
| Mean expression score (max 300) | 22.44 | 2.82 | 17.26 | 0.004 |
| SDHB | ||||
| Rate of global loss (%) | 0 | 0 | 0 | N/A |
| pS6 | ||||
| Mean expression score (max 300) | 26.59 | 72.89 | 60.23 | 0.001 |
ACC, adrenal cortical carcinoma; ACA, adrenal cortical adenoma; N/A, not applicable
scores were 203.24 in ACC versus 156.56 in ACAs versus 100.20 in normal adrenal cortical tissue samples. Cytoplasmic expression of PKM2 was significantly higher in ACC than in ACA (p =0.014) or normal adrenal cortical tissue samples (p <0.001) (Fig. 2). Expression of PKM2 was also higher in ACA than in normal adrenal cortical tissue samples (p < 0.001). In contrast, PKM1 expression was overall low in ACC, ACA, and normal adrenal cortical tissue samples. Intermediate-to-high intensity cytoplasmic PKM1 expression was seen in 7% of ACC versus 0% of ACA versus 13% of normal adrenal cortical tissue samples, while the mean PKM1 expression scores were 22.44 in ACC versus 2.82 in ACAs versus 17.26 in normal adrenal cortical tissue samples.
Cytoplasmic expression of PKM1 was higher in ACC than in ACA (p =0.027) but not statistically different when com- pared with normal adrenal cortical tissue samples (p =0.85) (Fig. 3). Significantly, HK1 expression correlated with pyru- vate kinase M2 and M1 isoforms (PKM2 and PKM1) expres- sion (r=0.5; p <0.001)(r=0.2; p =0.007), with no associa- tion seen between PKM2 and PKM1 expression (p = 0.28).
Expression Profile of Succinate Dehydrogenase
None of the 40 ACCs (2 of 42 ACCs technically failed) and 50 ACAs in our study cohort showed loss of succinate dehydro- genase (SDHB) expression by immunohistochemistry. Levels
a
b
c
d
e
f
a
b
C
d
e
f
were similar in most tumors with the exception of a small number of neoplasms enriched in oncocytic cells that showed more intense reactivity.
Expression Profile of PI3k Pathway-Related Phospho-S6 Ribosomal Protein
Intermediate-to-high intensity cytoplasmic phospho-S6 ribo- somal protein (pS6) expression was seen in 10% of ACC versus 34% of ACA versus 29% of normal adrenal cortical tissue samples (p <0.001). The mean pS6 expression scores were 26.59 in ACC versus 72.89 in ACAs versus 60.23 in normal adrenal cortical tissue samples (Table 2). Cytoplasmic expression of pS6 was lower in ACC than in ACA (p =0.003) or normal adrenal cortical tissue samples (p =0.008), while no difference in pS6 expression was seen between ACA and normal tissue samples (p =0.60) (Fig. 3). Expression of pS6 did not correlate with HK1, PKM2, and PKM1 expression.
Correlation of Metabolic Biomarkers with Tumor Mitotic Grade and Ki67 Labeling Index in Adrenal Cortical Carcinomas
Although none of the metabolic biomarkers correlated with the tumor mitotic grade (low grade vs high grade; based on the cutoff of 20 per 50 high power fields), HK1 expression was associated with Ki67 tumor proliferation index (r = 0.34; p = 0.03). None of the other metabolic biomarkers were associated with Ki67 tumor proliferation index.
ACA (b), and adrenal cortical tissue samples (c). Cytoplasmic expression of phosphorylated S6 ribosomal protein (pS6), a downstream target of the PI3k-AKT-mTOR pathway, was not overexpressed in ACC (d) compared with ACA (e) and normal adrenal cortical tissue samples (f)
Discussion
Metabolic reprogramming is a phenomenon with well- recognized contribution to carcinogenesis yet it remains poor- ly understood in adrenal cortical carcinoma [2, 8-11, 17, 18]. We characterized the metabolic phenotype of a large series of ACC, ACA, and normal adrenal cortical tissue samples, and demonstrated that HK1 and PKM2, two important glycolytic enzymes, are differentially expressed in malignant versus be- nign adrenal cortical tumors, as well as in neoplastic versus non-neoplastic adrenal cortical tissue.
Glycolysis is a central pathway of cell metabolism, in which glucose is converted to pyruvate through a series of reversible and irreversible enzymatic reactions [11, 14]. The energy derived from this process is used to generate ATP molecules, a process that is essential for mammalian life [11, 14]. In normal tissue, pyruvate is usually metabolized through the citric acid cycle when oxygen is present, and alternatively ferments to lactate when oxygen is lacking [11, 14]. A hall- mark of cancer cells is an increase in glucose consumption followed by accelerated fermentation to lactate, even in the presence of oxygen and fully functioning mitochondria [11, 14, 20]. This metabolic reprogramming phenomenon, known as aerobic glycolysis or the “Warburg effect,” is thought to be a required step to fulfill the anabolic demands of proliferating tumor cells, and has been described in various malignancies, including glioblastoma, colorectal cancer, gastric cancer, pan- creatic cancer, and breast cancer [11, 14, 21-25]. Prior imag- ing data using positron emission tomography (PET) and glu- cose analogue tracer 18fluorodeoxyglucose (FDG) have
shown a high level of glucose consumption in adrenal cortical carcinoma [8, 9, 26-28]. As a result, several groups have recently scrutinized the role of altered glucose metabolism in ACC pathogenesis and found that the glucose transporter (GLUT1) and monocarboxylate transporters (MCT4), two membranous transporters involved in glycolysis, are overexpressed in ACC when compared with ACA [8-10]. Glucose transporters are thought to play a crucial role in ac- celerated glycolysis, as cancer cells are only able to depend on the glycolytic pathway for energy production if the demands for increased glucose uptake by cancer cells are met [8, 15, 19]. Similarly, monocarboxylate transporters are also thought to be important for the “Warburg effect” as they are responsi- ble for lactate transport outside the cell to drive the glycolysis reaction forward [8, 10, 14]. Both Fenske et al. and Pinheiro et al. found higher GLUT1 expression in ACC compared with ACA [8, 10]. Similarly, MCT4 expression was higher in ACC compared with ACA [8]. Taken together, these findings sug- gest that metabolic reprogramming to a hyperglycolytic phe- notype may occur in a subset of ACC through overexpression of membrane transporters involved in glycolysis.
However, the contribution of intracellular glycolytic en- zymes in metabolic reprogramming of ACC remains unclear. A recent proteomics study revealed that aldolase A, an en- zyme responsible for a reversible step in glycolysis, is overexpressed in malignant compared with benign adrenal cortical tumors [17]. Our study findings add to the growing evidence that intracellular glycolytic enzymes may play a role in ACC pathogenesis, by demonstrating that HK1 and PKM2 are also differentially expressed in malignant versus benign adrenal cortical tumors, as well as in neoplastic versus non- neoplastic adrenal cortical tissue. Hexokinase catalyzes the first irreversible step of glycolysis, phosphorylating glucose to produce glucose-6-phosphate (G6P) [14]. This reaction is an important step in glucose energy metabolism because it traps glucose within cells by converting it to G6P, a conver- gence point of many important metabolic pathways including the pentose phosphate pathway (PPP), the hexosamine path- way, and glycogenesis [14]. By binding to the outer mitochon- drial membrane, hexokinase preferentially use ATP derived from mitochondria to phosphorylate glucose, thereby cou- pling oxidative phosphorylation (OXPHO) with glycolysis [14, 29]. Cancer cells often facilitate this first irreversible step of glycolysis by inducing high level expression of hexokinase [14], and this was also observed in our study, as HK1 expres- sion was significantly higher in ACC than in ACA or normal adrenal cortical tissue, suggesting perhaps that the first irre- versible step of glycolysis may be aberrantly activated in a subset of ACC. In addition to hexokinase, we found that py- ruvate kinase isoform M2 (PKM2) expression was also higher in ACC than in ACA or normal adrenal cortical tissue. Pyruvate kinase catalyzes the third and final irreversible step of glycolysis, converting phosphoenolpyruvate (PEP) into
pyruvate [14]. Contrary to other steps, the last committed step is attenuated in cancer cells, allowing metabolites to be diverted into branching pathways, such as the PPP and the serine biosynthesis pathway to generate sufficient metabolic intermediates and augment the anabolic reactions required for tumor growth and proliferation [14]. This attenuation can be achieved by favoring the use of low-affinity PKM2 to catalyze this reaction instead of the high-affinity PKM1 [14, 30], and our study found PKM2 expression to be significantly higher in ACC than in ACA or normal adrenal cortical tissue. Furthermore, PKM2 expression was associated with HK1 ex- pression in ACC, suggesting perhaps that metabolic remodel- ing towards a hyperglycolytic immunophenotype may occur in a subset of ACC through overexpression of intracellular enzymes involved in glycolysis.
In addition to the glycolysis pathway, we also examined the expression of the succinate dehydrogenase (SDH) complex in adrenal cortical tumors. The SDH complex plays a pivotal role in cellular energy metabolism, as it links the Krebs cycle to oxidative phosphorylation [18, 31]. Loss-of-function of SDH is thought to interrupt the Krebs cycle, leading to a state of cellular pseudohypoxia, which is described as one the most common causes of hereditary endocrine tumors [14, 18, 31-33]. While SDH mutations have not been widely implicat- ed in adrenal cortical tumorigenesis, a recent study examined the rare occurrence of SDH mutations in 4 unrelated individ- uals with ACC and germline SDHx mutations [18]. In this study, we evaluated the immunohistochemical expression of SDHB and found that none of the adrenal cortical tumors in our cohort showed loss of SDHB expression, suggesting that SDH loss-of-function may not be a salient feature of adrenal cortical neoplasia. We also used SDHB expression as a surro- gate biomarker of SDHx-related hypoxia and found no alter- ation in expression levels in the various tissues examined.
Recently, the metabolism of cancer cells has been the focus of much attention with the realization that many oncogenic drivers, including the PI3k-AKT-mTOR (mTOR) pathway, modify glucose metabolism [14]. A transgenic animal model of ACC showed that mTOR activation is an early step in tumorigenesis [34]. Furthermore, the TCGA data identified singleton gene fusions (EXOSC10-MTOR fusion) in ACC correlating with high phospho-mTOR levels [2]. Variable ex- pression of HK1, PKM, and RPS6 is also seen in the TCGA data of ACCs [2]. Nevertheless, the contribution of mTOR signaling to adrenal cortical carcinogenesis remains controversial [7, 35, 36]. A previous study by our group showed that adrenal cortical carcinomas and adenomas were no different with respect to PTEN and phospho-mTOR immunoexpression scores [7]. Similarly, De Martino and col- leagues did not find any difference in mRNA levels of mTOR between ACC and ACA [35]. In this study, we found that the expression of phosphorylated S6 ribosomal protein (pS6), a downstream target of the PI3k-AKT-mTOR pathway, was not
overexpressed in ACC compared with ACA and normal ad- renal cortical tissue. Similar findings were previously reported [35], suggesting that aberrant mTOR activation may be infre- quent in ACC.
Conclusion
Variable expression of HK1, PKM, and RPS6 was noted in the TCGA data of ACCs [2]. Our study provides further evidence that intracellular glycolytic enzymes, notably HK1 and PKM2, are differentially expressed in malignant versus be- nign adrenal cortical tumors, as well as in neoplastic versus non-neoplastic adrenal cortical tissue. Furthermore, our find- ing of glycolytic enzyme overexpression in ACC, which was largely unrelated to proliferative variables, adds to the grow- ing evidence that metabolic remodeling towards a hyperglycolytic phenotype may occur in a subset of ACC, similar to that described in other types of cancer. The possi- bility of utilizing the reprogrammed glucose metabolism in ACC as a potential tumor biomarker or for novel therapeutic strategies should be explored in future studies.
Acknowledgements The authors would like to thank both Drs. Thomas Giordano and Antonio Marcondes Lerario (University of Michigan Hospital, Michigan Medicine), for assisting the authors to obtain the expression status of HK1, PKM and RPS6 from the TCGA database.
Funding Information This work was funded in part by a Canadian Institutes of Health Research (CIHR) New Investigator Foundation Grant FDN-148385 (D.A.W.). D.A.W. holds an Ontario Ministry of Innovation Early Researcher Award.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
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