According to the U.S. Centers for Disease Control and Detection (CDC), colorectal cancer (CRC) was the third leading cause of cancer-related deaths in the United States in 2021 [1
]. Prognosis is partly dependent on stage at presentation according to the TNM classification system based on the American Joint Committee on Cancer (AJCC) [2
] and the Union for International Cancer Control (UICC) [3
]. Additional clinicopathologic variables can also impact survival, such as primary tumor location [4
], body mass index (BMI) [6
], and diabetes conditions [7
The application of biomarkers in a clinical context has the potential to improve prognosis based on gene mutations [8
], microRNAs [10
], and DNA methylation [11
]. During the last decade, cancer studies have benefited from the implementation of metabolomics [13
]. CRC metabolomic studies have shown the correlation of certain metabolite profiles to cancer survival and recurrence using biological samples of urine and tissue [14
]. Some plasma metabolites are associated with recurrence in stage II and III patients [15
], showing the potential value of metabolomics in finding prognostic biomarkers of CRC.
In addition, sex-related differences in CRC prognosis have become increasingly important in cancer research [16
]. Female patients have a higher prevalence of right-sided colon cancer (RCC), which is associated with poorer overall survival (OS) [18
]. However, male CRC patients have a survival disadvantage in most subgroups by anatomic location and age [20
]. At present, female and male patients do not receive different sex-specific therapies.
Previously, our untargeted metabolomics analyses in a large series of CRC tumor tissues revealed sex-specific metabolic subphenotypes that could influence tumor progression and response to therapeutics [21
]. Important metabolic pathways in these subphenotypes included asparagine synthesis, energy production (glycolysis and pentose phosphate pathway (PPP)), lysophospholipid synthesis, methionine metabolism, and polyamine synthesis [21
]. Particularly, female patients with RCC showed increased asparagine levels [21
], which can be produced intracellularly by an ATP- and glutamine-dependent reaction catalyzed by asparagine synthetase (ASNS) [22
]. By examining The Cancer Genome Atlas (TCGA) data, we discovered that high ASNS
expression was associated with poorer OS for female CRC patients [21
]. Additionally, increased ASNS
expression has been linked to aggressive tumors and poorer prognosis in other cancer types [23
]. These findings suggest that sex plays a vital role in CRC prognosis together with the covariates of metabolism and anatomic tumor location. A clear understanding of these sex-specific metabolic differences represents a golden opportunity to prolong the survival of CRC patients.
In this study, we examine whether there is a link between CRC tumor metabolites and prognosis stratified by sex of the patient. This is the first study to reveal that sex-related differences exist in primary tumor metabolite profiles that are associated with OS and recurrence-free survival (RFS) in patients who have undergone curative intent colectomy. We also identify metabolic pathways which are associated with poor survival only in female CRC.
To our knowledge, this is the first study to show that sex-specific differences in the CRC tumor metabolome are linked to prognosis based on data from one of the largest metabolomics study cohorts. It is also the first application of a novel approach to examine metabolic pathways and their associations to CRC prognosis. By applying state-of-the-art mass spectrometry to fresh frozen specimens acquired operatively in a large colorectal biorepository, we show that females have a distinct metabolite profile, underscored by asparagine metabolism, which correlates with prognosis after surgical colectomy. This suggests that females and males have unique colorectal tumor metabolism, which contributes to sex-specific clinical outcomes.
Our study revealed that 11 tumor metabolites were associated with CRC 5-year OS and five metabolites for 5-year RFS, and these associations were sex-specific. Asparagine and serine were associated with both 5-year OS and 5-year RFS in a sex-specific manner. Further examination of asparagine within the de novo asparagine synthesis pathway (catalyzed by ASNS), showed consistently poor OS and RFS with high asparagine abundance for females. This supports previous findings revealing poor female survival and high ASNS
expression from the TCGA [21
]. In addition, we previously hypothesized that asparagine is linked to tumor aggressiveness in females with RCCs as it correlates with increased threonine and serine uptake, which indicates aggressive tumor phenotypes [21
]. ASNS-catalyzed asparagine production has also been shown to be crucial for in vitro
cancer cell proliferation by amino acid homeostasis [25
], and the availability of asparagine in vitro and in vivo propagates the metastatic progression of breast cancer [26
In CRC, SRY-box 12 (SOX12
) expression promotes cell proliferation and metastasis, and it facilitates ASNS
]. In addition, expression of the mutant KRAS
gene correlates with a marked decrease in aspartate levels and increased asparagine levels due to upregulated ASNS
expression, which indicated that ASNS might be a novel therapeutic target for KRAS
-mutant CRC [28
]. Moreover, SLC25A22 serves as an essential metabolic regulator for CRC progression [30
] by promoting the synthesis of aspartate-derived amino acids (asparagine) in KRAS
-mutant CRC cells [31
]. In our study, KRAS
information was incomplete but further analysis on patients with complete KRAS
records can be found in Appendix B
and Table A2
), where asparagine showed similar sex-specific associations with both OS and RFS. Furthermore, increased dietary asparagine in animals promotes metastatic progression in breast cancer, and L-asparaginase treatment or dietary asparagine restriction inhibits metastasis [32
]. Hence, both internally produced asparagine and external exposure to asparagine through diet may influence CRC tumor progression. However, a review study pointed out that some studies are contradictory regarding whether ASNS
expression favors or inhibits CRC progression [33
], while our findings indicate that such inconsistency may attribute to sex differences.
As mentioned, serine was associated with both 5-year OS and RFS in a sex-specific manner. Our data showed that serine favored longer OS and RFS in males but was independent of prognosis in females. Serine metabolism has been reported to contribute to CRC metabolism and growth [34
] and enzymes involved in serine synthesis such as phosphoglycerate dehydrogenase (PHGDH) [35
] are found in higher levels in colonic tumor tissue than paired normal tissue [36
]. These studies did not consider sex differences that might lend to a potential sex-specific phenomenon.
Our results indicated that sex significantly modified the associations between lysophospholipid synthesis and CRC prognosis. LysoPC (16:0) was associated with worse OS for females in both individual metabolite analysis and the model for lysophospholipid synthesis. Our previous study identified that multiple LysoPCs and LysoPEs were upregulated in female patients with RCC (stage I) only, suggesting that the higher level of lysophospholipids in women with RCC would promote fatty acid supply that is essential for cancer cell growth at early stages [21
]. It has also been discussed that sex-specific estrogen regulation may inhibit CRC cell survival by suppressing triglyceride biosynthesis, a vital lipid marker related to CRC progression [17
Lastly, sex interacts with the associations between polyamine metabolism and CRC prognosis. Arginine and citrulline were associated with worse RFS and OS, respectively, for females but not for males. A previous study investigated the L-arginine/nitric oxide (NO) pathway in CRC by clinical stage, location of a primary tumor, and sex, and they observed elevated arginine levels in LCCs compared with RCCs, and a drop in arginine levels in the early postoperative period in females exclusively [37
]. Arginine and citrulline are important parts of the urea cycle [38
], where ornithine is produced and metabolized into polyamines by ornithine decarboxylase (ODC) [39
]. ODC is a target for inhibition by difluoromethylornithine (DFMO) (an anti-polyamine therapy for colorectal neoplasia) [40
], therefore, some of the polyamine precursors could be important for polyamine treatment responses, where sex can play a role. Moreover, there are NCI-funded cooperative group CRC chemoprevention trials targeting polyamine synthesis (PACES/SWOG) [41
], and a review study showed that methionine dietary restriction can suppress tumor formation, potentially through reducing the availability of decarboxylated SAM which is a cofactor for spermidine (a polyamine) production [42
]. Therefore, key metabolic pathways may have great applicability in sex-specific treatment or preventative interventions for CRC.
Although all female patients in our analysis were over 55 years and sex hormones might not be able to alter the associations between tumor metabolome and CRC prognosis, the observed sex differences could be attributed to other factors. Sex differences in metabolism are known to exist throughout life-course, as reflected by changes in amino acids level at birth [43
], fluctuation of lipid profile of males before and after puberty, with larger sex differences at older ages [44
]. Sex chromosomes such as the gene forkhead box P3 (FOXP3
) located on the X chromosome have been linked to CRC [45
]. In addition, there is evidence that sex differences exist before puberty when sex hormone levels rise, and life-course exposure to estradiol can result in elevated DNA and CpG island methylation, as well as demethylation which could result in genetic mutations at later life [46
]. Life-course exposure to sex hormones can alter microbiome diversity which would have an impact on microbial metabolism in the colon [47
]. Sex differences in the tumor microenvironment such as hypoxia, drug metabolism, and angiogenesis have been shown to impact sex-specific immune responses and oncogenes in CRC [17
Some of the study limitations include the number of events in the cohorts compared to sample size, and the multiple categories such as clinical stage, sex group, anatomic location, which might weaken statistical power. In addition, prognostic factors such as KRAS
] and BRAF
mutational status [49
], and microsatellite instability status [50
] were incomplete in our data; thus, they were not considered in survival analyses for metabolic pathways to avoid less statistical power. Therefore, future studies from an independent cohort with more comprehensive molecular profiling will be valuable. Further, metabolomic analysis of normal colorectal tissues that surround the tumor would also enhance the current findings.
In conclusion, we apply a novel methodology using tumor tissue to identify sex-specific metabolic pathways associated with CRC prognosis in a surgical cohort. We show that tumor metabolite levels have different associations with patient prognosis by sex, in particular ASNS-catalyzed asparagine production that is linked to poor prognosis only in females. Our study shows that sex-specific tumor metabolism is an important factor for understanding differences in patient prognosis and potentially differences in treatment responses. Accordingly, we recommend that the design of precision medicine approaches consider both tumor metabolism and sex to obtain more robust findings in clinical trials and improve patient outcomes.