Tumor Biology

, Volume 35, Issue 7, pp 6823–6830 | Cite as

Deficiency in asparagine synthetase expression in rectal cancers receiving concurrent chemoradiotherapy: negative prognostic impact and therapeutic relevance

  • Ching-Yih Lin
  • Ming-Jen Sheu
  • Chien-Feng Li
  • Sung-Wei Lee
  • Li-Ching Lin
  • Yi-Fong Wang
  • Shang-Hung Chen
Research Article

Abstract

Locally advanced rectal cancers are currently treated with neoadjuvant concurrent chemoradiotherapy (CCRT) followed by surgery, but risk stratification and final outcomes remain suboptimal. In this study, we identify and validate targetable metabolic drivers relevant to the prognosis of patients with rectal cancer treated with CCRT. Using a published transcriptome of rectal cancers, we found that asparagine synthetase (ASNS) gene significantly predicted the response to CCRT. From 172 patients with rectal cancer, the expression levels of ASNS, using immunohistochemistry assays, were further evaluated in tumor specimens initially obtained by using colonoscopy. Expression levels of ASNS were further correlated with major clinicopathological features and clinical survivals in this valid cohort. ASNS deficiency was significantly related to advanced posttreatment tumor (T3, T4; P = .015) and nodal status (N1, N2; P = .004) and inferior tumor regression grade (P < .001). In survival analyses, ASNS deficiency was significantly associated with shorter local recurrence-free survival (LRFS; P = .0039), metastasis-free survival (MeFS; P = .0001), and disease-specific survival (DSS; P = .0006). Furthermore, ASNS deficiency was independently predictive of worse outcomes for MeFS (P = .012, hazard ratio = 3.691) and DSS (P = .022, hazard ratio = 2.845), using multivariate analysis. ASNS deficiency is correlated with poor therapeutic response and worse survivals in patients with rectal cancer receiving neoadjuvant CCRT. These findings indicate that ASNS is a prognostic factor with therapeutic potential for treating rectal cancer.

Keywords

ASNS Rectal Cancer CCRT 

Abbreviations

CCRT

Chemoradiotherapy

ASNS

Asparagine synthetase

LRFS

Local recurrence-free survival

MeFS

Metastasis-free survival

DSS

Disease-specific survival

LARC

Locally advanced rectal cancer

ALL

Acute lymphoblastic leukemia

Pre-Tx

Pre-treatment

Post-Tx

Post-treatment

AJCC

American Joint Committee on Cancer

TRG

Tumor regression grade

HR

Hazard ratio

PAH

Phenylalanine hydroxylase

HCC

Hepatocellular carcinoma

NPC

Nasopharyngeal carcinoma

Introduction

Rectal cancer, defined as a cancerous lesion of the colon distal to the rectosigmoid junction, is an increasingly common disease in Taiwan, with an estimated 5,400 new cases per year in 2010 [1]. Combined modality therapy, consisting of surgery, radiotherapy, and chemotherapy, is recommended for the majority of patients with stage II or stage III rectal cancer. Randomized phase III trials have demonstrated that neoadjuvant concurrent chemoradiotherapy (CCRT) for locally advanced rectal cancer (LARC) (Stage T3, T4, or node-positive disease) significantly improves local control and reduces toxicity profiles, compared with adjuvant CCRT [2, 3, 4]. Furthermore, recent studies indicated that pathologic downstaging, or a complete pathologic response, following neoadjuvant CCRT is correlated with improved survival and a higher rate of sphincter-preserving surgeries [5, 6, 7]. Currently, neoadjuvant CCRT followed by surgery is the standard treatment for LARC. Although outcomes using this approach are encouraging, only 40–60 % of patients with LARC treated with neoadjuvant CCRT achieve some degree of pathologic downstaging [4, 8]; this leaves substantial room for improvement in survival outcomes for such patients. Developing potential therapeutic targets is a key to provide options for clinical management of patients with LARC.

Various genetic, epigenetic, and posttranslational aberrations of metabolism-associated enzymes may play essential roles in malignant transformation and sustained tumor cell growth [9, 10, 11]. Therefore, identifying specific alterations in metabolic pathways may generate opportunities for designing new therapeutic approaches. As tumors grow, high levels of exogenous essential and nonessential amino acids are required to sustain higher proliferative rates and resist some cell death signals [9, 10, 11, 12, 13]. These alterations in metabolic pathways generate opportunities to develop compounds targeting amino acid metabolism in cancer therapy. In order to investigate novel targets of amino acid metabolism in LARC, we analyzed the published transcriptome of patients with rectal cancer (GSE35452) and identified the asparagine synthetase (ASNS) gene as a determinant of response to neoadjuvant CCRT. ASNS encodes the enzyme that catalyzes l-aspartate and l-glutamine into l-asparagine and l-glutamate in an ATP-dependent reaction [14]; the transcription of the ASNS gene is highly regulated in response to cell stress [15]. The clinical effects of plasma asparagine depletion using l-asparaginase have been determined in acute lymphoblastic leukemia (ALL) [16, 17, 18]. However, the potential relationship between ASNS expression and its clinical relevance in human malignancies, including rectal cancers, has not been extensively studied. In this study, we evaluate the clinical implications of ASNS expression in a well-defined cohort of 172 patients with LARC treated with neoadjuvant CCRT followed by surgery.

Materials and methods

Analysis of published transcriptomic data sets

In order to investigate genes critical in the response to neoadjuvant CCRT, we reappraised one public transcriptome of tissues from rectal cancers (n = 46). The expression profiling data set deposited in the Gene Expression Omnibus had the accession number GSE35452. We imported the raw CEL files of Affymetrix HUMAN Genome U133 Plus 2.0 microarray platform into Nexus Expression 3 software (BioDiscovery) to analyze all probe sets without preselection. Comparative analysis and functional profiling were performed to identify significant differentially expressed genes, with special attention to pathways involving amino acid biosynthesis in Gene Ontology (GO: 0008652). These gene candidates with P < .01 and log2-transformed expression fold change >0.1 were chosen for further analysis.

Patient eligibility and follow-up

The institutional review board approved procurement of formalin-fixed tissue of patients with rectal cancer for this study (IRB 10302-014). We retrieved a total of 172 patients with rectal cancer with paraffin-embedded tissue blocks and periodic follow-up from the archive of Chi Mei Medical Center between 1998 and 2004. At initial presentation, these patients were confirmed as having an adenocarcinoma of the rectum using a colonoscopic biopsy, and no distant metastasis by chest X-radiography and/or abdominopelvic CT. For all 172 patients with rectal cancer, radiation therapy was given at a total dose of 45 Gy in 25 fractions over a period of 5 weeks with 24-h continuous infusion of 5-fluorouracil concurrently before surgery. Adjuvant systemic chemotherapy was administered if the pretreatment (pre-Tx) or posttreatment (post-Tx) stage of the tumor was beyond T3 or N1. All patients were regularly monitored after diagnosis until death or their last appointment.

Histopathologic evaluation

Two independent pathologists who had not been given the patients’ clinical information performed pathologic analyses of the tumor specimens. Post-Tx T and N stages of all patients were documented according to the 7th American Joint Committee on Cancer (AJCC) TNM staging system [19]. They reviewed hematoxylin–eosin-stained sections and evaluated proximal, distal, and circumferential resection margins. A careful search of the mesorectum was performed to identify as many lymph nodes as possible. Tumor regression grades (TRGs), used as end points for evaluation of tumor response after neoadjuvant CCRT, were documented as previously described [20, 21].

ASNS immunohistochemistry and scoring

Tissue sections from pre-Tx rectal tumor biopsies were cut from paraffin-embedded tissue blocks at 3-mm thickness onto precoated slides. For ASNS immunostaining, the slides were deparaffinized with xylene, rehydrated with ethanol, and heated by microwave treatment for retrieval of antigen epitopes in a 10-mM citrate buffer (pH 6) for 7 min. Endogenous peroxidase was quenched by 3 % H2O2 treatment. The slides were washed with Tris-buffered saline for 15 min and then incubated with a primary monoclonal antibody against ASNS (1:50; Epitomics catalog number 1732-1). In an attempt to accurately describe the extent of immunohistochemical staining of a tumor, the ASNS staining was interpreted as a continuous variable using the H-score [12, 13]. The intensity of the staining was scored according to four categories: 0 for “no staining”; 1+ for “light staining, visible only at high magnification”; 2+ for “intermediate staining”; and 3+ for “strong staining, visible even at low magnification” as seen in Fig. 2. The percentage of cells at different staining intensities was determined by visual assessment, with the H-score calculated using the formula 2 × (the percentage of 1+ cells) + 3 × (the percentage of 2+ cells) + 4 × (the percentage of 3+ cells). Tumors with H-scores less than the median of all scored cases were classified as having low ASNS expression.

Statistical analysis

SPSS 14 software package was used for statistical analyses. The comparisons of ASNS expression between subgroups defined by various clinicopathological parameters were evaluated by chi-square test. Local recurrence-free survival (LRFS), metastasis-free survival (MeFS), and disease-specific survival (DSS), calculated from the date of operation to the date of event, were the endpoints analyzed. We plotted survival curves using the Kaplan–Meier method and performed log-rank tests to evaluate prognostic differences between groups. Multivariate analysis was performed using the Cox proportional hazard model. For all analyses, two-sided tests of significance were used, with P < .05 considered significant.

Results

Downregulation of ASNS gene predicts non-responders to CCRT

From the data set of 46 rectal cancer cases in the public transcriptome GSE35452, we focused on 30 probes covering 22 genes regulating amino-acid biosynthesis. In non-responders to CCRT, phenylalanine hydroxylase (PAH) and ASNS displayed significantly downregulated mRNA expression (P < .01, Fig. 1, Table 1). Of these, downregulation of ASNS, but not PAH, possesses a unique therapeutic relevance. This finding prompted us to further characterize the expression status and clinical relevance of ASNS in rectal cancers.
Fig. 1

Analysis of ASNS expression in responders versus non-responders to CCRT from a published transcriptomic data set of rectal cancers. In the clustering analysis of gene regulating amino acid biosynthesis, ASNS was significantly downregulated in non-responders to CCRT. Tissue specimens from non-responders (blue lines) and responders (yellow lines) are indicated on top of the heat map, and expression levels of upregulated and downregulated genes are expressed as a spectrum of brightness of red and green, respectively, with those unaltered in mRNA expression being coded as black

Table 1

Summary of differentially expressed genes associated with amino acid biosynthesis (GO: 0008652) in rectal carcinoma in relation to response to CCRT

Probe

Comparison log ratio

Comparison p value

Gene symbol

Gene name

Biological process

Molecular function

205047_s_at

−0.5811

0.0047

ASNS

Asparagine synthetase

Amino acid biosynthetic process, asparagine biosynthetic process, glutamine metabolic process, and metabolic process

Asparagine synthetase (glutamine-hydrolyzing) activity, and ligase activity

205719_s_at

−0.7284

0.0016

PAH

Phenylalanine hydroxylase

l-Phenylalanine catabolic process, amino acid biosynthetic process, aromatic amino acid family metabolic process, and metabolic process

Amino acid binding, catalytic activity, iron ion binding, metal ion binding, monooxygenase activity, oxidoreductase activity, and phenylalanine 4-monooxygenase activity

Immunohistochemical expression of ASNS and its association with clinicopathological features

To further investigate the correlation between the expression of ASNS and its clinical relevance in rectal cancers treated with neoadjuvant CCRT, we first examined the expression of ASNS in clinical specimens using immunohistochemistry. When detected in cell cytoplasm, ASNS immunoexpression was successfully scored in all 172 cases with a wide range of H-scores, varying from 105 to 380 (Fig. 2). As shown in Table 2, low expression of ASNS was correlated with an advanced post-Tx tumor status (P = .015) and nodal status (P = .004), respectively. Furthermore, higher TRG, meaning better response after CCRT in rectal cancer, has been proved closely related with better survival and low local recurrence [20, 21]. As expected, low expression of ASNS was positively correlated with the inferior TRG degree (P < .001, Fig. 2), suggesting the biological role of ASNS in modulating the sensitivity of rectal cancers to CCRT. However, the ASNS expression levels were not statistically related to other clinicopathological variables. Pre-Tx and post-Tx characteristics of all patients are summarized in Table 2.
Fig. 2

Representative immunostainings of ASNS expression in rectal cancers. High expression (a) and low expression (b) of ASNS in pretreatment specimens demonstrated were linked to remarkable tumor regression (c) and low tumor regression grade (d) after CCRT, respectively. Original magnification ×200

Table 2

Associations and comparisons between ASNS expression and clinicopathological factors in 172 patients with rectal cancer receiving neoadjuvant CCRT

Parameter

 

No.

ASNS expression

p value

High expression

Low expression

Gender

Male

108

55

53

0.752

Female

64

31

33

 

Age

<70

106

53

53

1.000

≧70

66

33

23

 

Pre-Tx tumor status (pre-T)

T1–T2

81

39

42

0.647

T3–T4

91

47

44

 

Pre-Tx nodal status (pre-N)

N0

125

67

58

0.124

N1–N2

47

19

28

 

Post-Tx tumor status (post-T)

T1–T2

86

51

35

0.015*

T3–T4

86

35

51

 

Post-Tx nodal status (post-N)

N0

123

70

53

0.004*

N1–N2

49

16

33

 

Vascular invasion

Absent

157

79

78

0.787

Present

15

7

8

 

Perineural invasion

Absent

167

84

83

0.650

Present

5

2

3

 

Tumor regression grade

Grade 0–1

37

4

33

<0.001*

Grade 2~3

118

67

51

 

Grade 4

17

15

2

 

*Statistically significant

Prognostic impact of ASNS expression in rectal cancer

Next, we analyzed the correlation between ASNS expression and the prognosis of patients with rectal cancer by Kaplan–Meier analysis. The mean follow-up period of these patients was 48.2 months (range 6.2 to 131.2). A number of clinicopathological parameters including the pre-Tx nodal status, post-Tx tumor status, post-Tx nodal status, vascular invasion, and TRG (Fig. 2) were predictive of at least one of the three endpoints of this study, according to univariate analysis (Table 3). Notably, low expression of ASNS also pursued a more aggressive clinical course, with significantly shorter DSS (P = .0006; Fig. 3), LRFS (P = .0039; Fig. 3), and MeFS (P = .0001; Fig. 3). In multivariate comparison, vascular invasion and post-Tx tumor status were also found to have at least one independent prognostic impact on survival. Most importantly, low expression of ASNS remained an independent prognostic factor for both DSS (P = .022, hazard ratio [HR] = 2.845) and MeFS (P = .012, HR = 3.691), while it lost statistical significance with respect to LRFS (Table 4).
Table 3

Univariate log-rank analysis of important clinicopathological variables and ASNS expression

Parameter

 

No. of cases

DSS

LRFS

MeFS

No. of events

p value

No. of events

p value

No. of events

p value

Gender

Male

108

20

0.9026

7

0.2250

17

0.3520

Female

64

11

 

20

 

14

 

Age

<70

106

19

0.8540

18

0.6615

20

0.7427

≧70

66

12

 

9

 

11

 

Pre-Tx tumor status (pre-T)

T1–T2

81

10

0.0776

10

0.2261

11

0.1745

T3–T4

91

21

 

17

 

20

 

Pre-Tx nodal status (pre-N)

N0

125

19

0.0711

15

0.0070*

19

0.0973

N1–N2

47

21

 

12

 

12

 

Post-Tx tumor status (post-T)

T1–T2

86

7

0.0006*

7

0.0040*

8

0.0033*

T3–T4

86

24

 

20

 

23

 

Post-Tx nodal status (post-N)

N0

123

21

0.5998

16

0.1320

20

0.4634

N1–N2

49

10

 

11

 

11

 

Vascular invasion

Absent

157

25

0.0184*

21

0.0028*

27

0.4470

Present

15

6

 

6

 

4

 

Perineural invasion

Absent

167

29

0.2559

25

0.0940

30

0.9083

Present

5

2

 

2

 

1

 

Tumor regression grade

Grade 0–1

37

13

0.0038*

10

0.0090*

14

0.0006*

Grade 2~3

118

17

 

17

 

16

 

Grade 4

17

1

 

0

 

1

 

ASNS expression

High expression

86

7

0.0006*

7

0.0039*

5

0.0001*

Low expression

86

24

 

20

 

26

 

DSS disease-specific survival, LRFS local recurrence-free survival, MeFS metastasis-free survival

*Statistically significant

Fig. 3

Kaplan–Meier survival curves plotted to predict survival. Using the log-rank test, low expression of ASNS predicted inferior disease-specific survival (a), local recurrence-free survival (b), and metastasis-free survival (c)

Table 4

Multivariate analysis

Parameter

DSS

LRFS

MeFS

HR

95 % CI

p value

HR

95 % CI

p value

HR

95 % CI

p value

Tumor regression grade

1.634

0.787–1.634

0.188

1.984

0.894–4.405

0.092

1.368

0.961–1.162

0.082

ASNS expression

2.845

1.161–6.967

0.022*

2.298

0.919–5.743

0.075

3.691

1.334–10.216

0.012*

Vascular invasion

2.715

1.075–6.855

0.035*

2.830

1.048–7.641

0.040*

 

 

Post-Tx tumor status (post-T)

2.645

1.103–6.340

0.029*

2.089

0.860–5.078

0.104

1.998

0.860–4.644

0.108

Pre-Tx nodal status (pre-N)

1.950

0.844–4.508

0.118

 

 

DSS disease-specific survival, LRFS local recurrence-free survival, MeFS metastasis-free survival

*Statistically significant

Discussion

Although neoadjuvant CCRT followed by surgery has become the standard treatment for patients with LARC, only 40–60 % of patients treated with neoadjuvant CCRT achieve some degree of pathologic downstaging, which has been correlated with improved survival [4, 8]. To better manage patients with LARC, it is necessary to develop more efficacious therapeutic strategies. Because cancers are genetic diseases, methods for identifying genetic biomarkers with significant potential for predicting treatment response that could lead to more individualized therapy are critical in modern cancer therapy. Genome-wide approaches with derived targeted therapies have prompted efforts to characterize coordinately regulated gene expressions of specific pathways [22]. Recently, metabolic alteration and adaptation of cancer cells have been extensively studied. It creates a phenotype that is essential for tumor cell growth and survival and offers a potential therapeutic target for cancer therapy. In a published transcriptome of rectal cancers, we first identified that downregulated ASNS gene within the pathways regulating amino acid metabolism was a significant candidate for predicting nonresponders to CCRT. Furthermore, for the first time, we identified ASNS deficiency that had negative prognostic implications for patients with rectal cancer receiving neoadjuvant CCRT. These findings may contribute to developing a new avenue of therapeutic strategies for LARC, since ASNS deficiency is known to confer preferential susceptibility to plasma asparagine depletion using l-asparaginase.

Because cells exhibit a wide spectrum of adaptive processes that serve to sense and respond to fluctuations in the environment, the expression of ASNS differs in various normal tissues and human cancers [23]. Recently, additional biological functions of ASNS other than amino acid synthesis have been delineated. First, ASNS has been substantiated in vitro and in vivo as a tumor suppressor in hepatocellular carcinoma (HCC) via its anti-proliferative, anti-migratory, and anti-invasive properties [24]. Low expression of ASNS also has been demonstrated to be correlated with worse survival outcomes in HCC. One possibility is that the deficiency in ASNS expression may lead to the accumulation of aspartate and glutamine, which are essential in the biosynthesis of purines and pyrimidines. Subsequently, the intracellular metabolism may shift to nucleic acid synthesis, therefore affecting cell proliferation. Furthermore, overexpression of ASNS has been proven to potentiate cisplatin-induced DNA damage and apoptosis in nasopharyngeal carcinoma (NPC) [25]. Ectopic overexpression of ASNS in NPC cells is reported to reduce the expression level of survival genes, including B-cell CLL/lymphoma 2, X-linked inhibitor of apoptosis, and baculoviral IAP repeat containing 5, as well as genes involving nucleotide excision repair, such as RAD23 homolog B and xeroderma pigmentosum, complementation group A. In contrast, knocking down of ASNS expression increases the expression of these genes involving cell survival and DNA repair. These characteristics as tumor suppressors or enhancers of DNA damage are possible mechanisms to clarify the prognostic impact of ASNS in rectal cancers.

l-Asparaginase, an enzyme that catalyzes the hydrolysis of asparagine to aspartic acid, has been widely used in chemotherapeutic protocols for treating ALL for almost 50 years [16, 17, 18]. Although the exact molecular basis underlying the therapeutic utility of l-asparaginase remains poorly defined, the death of leukemic cells induced by the depletion of cellular asparagine has been postulated for years [14, 26]. In solid tumors, recent studies have demonstrated that HCC and pancreatic and ovarian cancer cells with low ASNS expression are more sensitive to l-asparaginase than cells with high ASNS expression [24, 27, 28]. Based on the findings of other studies mentioned above as well as our own, ASNS expression might be a potential therapeutic target for the treatment of human cancers. Stratifying patients with rectal cancers on the basis of ASNS expression level is justified. Because ASNS deficiency has a negative impact on prognosis, studies are warranted to evaluate the effects of combination therapy of l-asparaginase and CCRT for rectal cancers with ASNS deficiency. In addition, small molecules capable of inhibiting cellular ASNS that function in a potent and highly selective manner have been recognized, including chemical constraints to the synthetase active site and sulfonamide derivatives as inhibitors [14]. The inhibition of ASNS expression together with l-asparaginase administration might be a useful strategy for the treatment of patients with rectal cancer with high ASNS expression.

PAH, another downregulated gene identified in the public transcriptome, encodes enzyme that catalyzes the hydroxylation of the aromatic side chain of phenylalanine to generate tyrosine. PAH deficiency, traditionally known as phenylketonuria, results in the accumulation of phenylalanine in the blood of affected individuals. Hyperphenylalaninemia can be toxic to brain and cognitive development; however, its clinical relevance in rectal cancers or other human malignancies has yet to be determined.

In conclusion, this is the first time that deficiency in ASNS expression is demonstrated to be significantly correlated with poor response to neoadjuvant CCRT for rectal cancer, including advanced post-Tx tumor and nodal status and lower grade TRG. More importantly, ASNS deficiency is a significant predictor of worse prognoses, especially DSS and MeFS, in patients with rectal cancer receiving neoadjuvant CCRT. These findings warrant further investigation into additional biological functions underlying this protein expression and combination strategies using l-asparaginase and/or ASNS inhibitors in rectal cancers.

Notes

Acknowledgments

This study is supported by the Chi Mei Medical Center (CMFHR10303 and CMNCKU10202) and Ministry of Health and Welfare (MOHW103-TD-B-111-05).

Conflicts of interest

None

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Ching-Yih Lin
    • 1
    • 2
  • Ming-Jen Sheu
    • 1
  • Chien-Feng Li
    • 3
    • 4
    • 5
    • 6
  • Sung-Wei Lee
    • 7
  • Li-Ching Lin
    • 8
  • Yi-Fong Wang
    • 2
  • Shang-Hung Chen
    • 4
    • 9
  1. 1.Division of Gastroenterology and Hepatology, Department of Internal MedicineChi Mei Foundation Medical CenterTainanTaiwan
  2. 2.Department of Leisure, Recreation, and Tourism ManagementSouthern Taiwan University of Science and TechnologyTainanTaiwan
  3. 3.Department of PathologyChi Mei Medical CenterTainanTaiwan
  4. 4.National Institute of Cancer ResearchNational Health Research InstitutesTainanTaiwan
  5. 5.Department of BiotechnologySouthern Taiwan University of Science and TechnologyTainanTaiwan
  6. 6.Institute of Clinical MedicineKaohsiung Medical UniversityKaohsiungTaiwan
  7. 7.Department of Radiation OncologyChi Mei Medical CenterTainanTaiwan
  8. 8.Department of Radiation OncologyChi Mei Medical CenterTainanTaiwan
  9. 9.Division of Hematology and Oncology, Department of Internal MedicineChi Mei Medical Center, LiouyingTainan CityTaiwan

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