Local, intestinal biomarkers
for early detection of
colorectal cancer
Master’s degree project in Drug Discovery and Development 30 HP, Spring 2021
Uppsala University, Department of Pharmacy Martina Andersson
Supervisors: Alexandra Teleki & Maria Karlgren Examinator: Per Artursson
Table of contents
Abstract ... 2
1 Introduction ... 2
2 Methods ... 4
2.1 Database search for mutations frequently observed in early onset of CRC ... 4
2.2 Database search for relevant preclinical in vitro and in vivo models representing early-stage CRC ... 5
2.3 Database search for studies performing global proteomics ... 5
2.4 Proteomics data analysis ... 5
2.5 Biomarker selection ... 6
3 Results ... 6
3.1 Typical mutations in early CRC ... 6
3.2 Cell lines representing early stages of CRC ... 7
3.3 Mouse models representing early stages of CRC ... 7
3.4 Protein expression in human tissue samples from early CRC ... 8
3.5 Biomarker Selection ... 9
4 Discussion ... 12
5 Conclusion ... 14
6 Future outlook ... 15
Abstract
Colorectal cancer (CRC) is one of the deadliest cancers in the world. The early stage of the disease is usually asymptomatic and therefore screening methods for colorectal cancer need to improve. There is a need for early detection of CRC as treatment is less effective in the advanced stage of the disease. The current standard screening methods are endoscopy and fecal immunochemical blood tests. Endoscopy is a commonly used method to diagnose the patient, but it is costly, time consuming, and rather unpopular for the patients. An alternative could be to develop targeted molecular imaging probes that specifically deliver agents for example magnetic resonance imaging to colon adenomas and adenocarcinomas. This alternative would be non-invasive and able to detect the disease before morphological changes become evident.
Biomarkers are used as an objective indicator of an altered biological process. Here, a literature study was conducted to identify protein biomarkers that are overexpressed in early stages of CRC. This study has focused on biomarkers that could be used to target imaging agents to cancerous lesions. Thus, the biomarkers need to be membrane-bound and expressed on the luminal side of the gastrointestinal tract. This will help future research to develop orally administered targeted imaging probes. Furthermore, a smaller literature search was conducted to identify cell and mouse models representing early stages of CRC. This was done to facilitate translational research going from in vitro to in vivo. Ideally the same protein is available in cell lines, mouse models and humans to enable translational research. This work has resulted in the selection of 7 different proteins that are upregulated during early stages of CRC. These proteins are potentially apically located and therefore possible targets for
monoclonal antibodies. These findings might lead to a novel way for preventive patient screening and hopefully reduce the mortality for colorectal cancer.
1 Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world and the second most reported for mortality (1). Both hereditary and environmental risk factors play a part in the development of CRC. Patients with long-standing inflammatory bowel disease (IBD) and those with a previous history of CRC or adenomas have an increased risk to develop colorectal cancer (2). The development of CRC is strongly influenced by the inflammatory state of the colon (2,3). Patients can show symptoms such as rectal bleeding, a
change in bowel habits, anemia or abdominal pain. However, colorectal cancer is largely asymptomatic until it reaches an advanced stage (2). The early stage of CRC is defined as the disease stage with adenoma where no surrounding tissues is affected, and no metastases. During the early stages of CRC, a patient screening can detect progressive adenomas that will eventually lead to colon carcinoma if untreated. It is during the episodes before carcinoma that early biomarkers might be present (4). The progress from adenoma to carcinoma is illustrated in Figure 1, the different stages of CRC can be divided into stage I-IV. Stage I-II are early stages where a tumor can be found but no nodes or metastases (5). Often it is
possible to treat CRC with removal of small benign colorectal adenomas, before they advance to malignancy. There is a need for early detection of CRC as the treatment is less effective in the advanced stage of the disease (6). An adenoma could take more than ten years to progress to cancer, this gives a good time window for screening (4). The current screening methods are optical colonoscopy or fecal immunochemical test (FIT) that detects occult bleeding. But the major issue with optical colonoscopy is that it is costly, invasive, and time-consuming (7). Computed tomography (CT) is used as a complementary imaging method for the diagnosis of polyps and colorectal cancer (2).
Figure 1. Schematic illustration of different stages in colorectal cancer inside the intestine and the way a monoclonal antibody can target apically located biomarkers. The red triangles represent a biomarker apically located that exists during early stages of CRC, as well as later stages. The green rectangular shape represents apically located proteins that exist on healthy normal epithelium, that are not of interest as CRC biomarkers.
A biomarker is an objective, quantifiable characteristic that can act as an indicator of a normal or altered biological process or activity (8). Finding biomarkers that reflect the early
epithelial cells of the colon change very early before histological changes become evident. For example, de Wit et al. (9) identified glucose transporter type 1 and prion protein as cell surface biomarkers in CRC. They investigated the adenoma to carcinoma progress in cell lines aiming to find biomarkers that could be targeted for molecular imaging (9). The analytical techniques used for proteomics can be for example mass spectrometry or chromatographic separation (10). Proteomics analysis is a powerful tool in biomarker
discovery and it can study the protein expression in cell lines, tissues or biological fluids (11). Cell surface proteins are attractive as biomarkers as they can be targeted by monoclonal antibodies (9). Biomarker targets could be identified to develop non-invasive, in situ imaging-based diagnostic tools. Molecular magnetic resonance imaging (MRI) uses contrast agents targeting overexpressed disease biomarkers to image disease location in vivo (11,12). With that in mind, identifying CRC biomarkers and developing targeted imaging probes that can be orally administered (Figure 1) (6) to the patients might represent an effective non-invasive pre-screening tool to guide personalized colonoscopy screening recommendations.
The aim of this project is to find apically located biomarkers in human intestinal mucosa tissue samples for the early stages of CRC. A literature study was conducted to identify clinical biomarkers for CRC. The search for biomarkers focused on studies performed on human tissues investigating early CRC. The data was analyzed to select membrane-bound proteins that are accessible from the luminal side of the colon. Furthermore, a smaller literature search was conducted so see what cell and mouse models are representative for early stages of CRC.
2 Methods
2.1 Database search for mutations frequently observed in early onset of CRC
First, a database search was conducted, aimed to find information regarding mutations that are relevant in the early stages of CRC (Figure 2, Step 1). Search was done using PubMed and the search terms are listed in Supplementary Table S1. The information obtained was summarized in Microsoft ® Excel (Version 16.48) and was used in section 2.2 to find relevant cell and mouse models for early CRC.
2.2 Database search for relevant preclinical in vitro and in vivo models representing early-stage CRC
A database search was conducted using PubMed to identify cell lines and mouse models that could represent early stages of CRC (Figure 2, Step 1). Search terms used can be found in supplementary table S1. Cell lines were investigated using information from the Swiss
bioinformatics resource portal Expasy (13) and ATCC (14). The cell lines investigated were summarized in Microsoft Ò Excel. The cell lines obtained during early stages of the disease were considered most relevant for this project. Both the cell lines and the mouse models should have mutations that can be frequently observed in early onset of CRC (section 2.1).
2.3 Database search for studies performing global proteomics
Database search for studies performing global proteomics was done in PubMed to find articles focusing on biomarkers representing early stages of CRC (Figure 2, Step 2). The search terms used are listed in Supplementary Table S1. The proteomics datasets found should only be from early stages. Articles selected were those that used human tissue samples of patients with early stages of CRC, together with healthy colon mucosa samples to compare the results. Some articles also included data from later stages. This data was used to
investigate whether the biomarker upregulates or downregulates as the disease progresses. Finally, biomarkers identified in the human tissue studies were compared with the data from cell lines and mouse models (section 2.2).
2.4 Proteomics data analysis
The goal of the proteomics’ data analysis was to find proteins that were upregulated with a fold change of > 1,5. The fold change is the ratio of the protein expression in cancerous tissue compared to normal tissue. In other studies for example fold change between 1,4-4 have been used (11,15,16). Here, a relatively low threshold for fold change of 1,5 was chosen to not miss any biomarkers but ultimately a high fold change for biomarkers is desirable. A criterion for significant biomarkers was also a p-value <0,05. When looking at the p-value, 2-sided student’s t-test was calculated using Microsoft ® Excel (Figure 2, Step 3a). In a first step, Human Protein Atlas (17) and UniProt (18) were used to determine the subcellular location of the biomarkers. The proteins that were stated as “plasma membrane” in their subcellular annotation were kept and listed using Microsoft ® Excel (Figure 2, Step 3b).
2.5 Biomarker selection
For the biomarker selection, proteins that were identified in at least two articles, were evaluated further (Figure 2, Step 4). A new database search was done with PubMed for these
proteins to find information if they are apically located. Search words used in combination with the protein names were apical; cell surface; membrane and plasma membrane. The proteins that might be apically located on the cell surface membrane were selected as candidate biomarkers (Figure 2, Step 5).
Figure 2. Overview of workflow to select apically located proteins relevant in early stages of colorectal cancer.
3 Results
3.1 Typical mutations in early CRC
The mutations Adenomatous polyposis coli (APC), Kirsten-Ras (KRAS) and Tumor protein p53 (TP53) are frequently observed during early stages of CRC and are summarized in supplementary Table S2. APC is a gene that can be detected mutated at an early stage of CRC (19–22). Loss of APC is followed by mutation in other genes including KRAS, TP53, PIK3CA and BRAF (21). APC also leads to hyperactivation of the Wnt signaling pathway (22,23). KRAS mutation is established in early in CRC and can be found about the same
levels in all stages of CRC (24–26). The most frequent KRAS mutation in stage I CRC occurs at codons 12 and 13 (G13D, G12D and 12V) (27). More than 50 % of patients with
adenocarcinoma do have a mutant allele of KRAS (24). TP53 is classified as an early mutation but can be found in both adenomas and carcinomas (19).
3.2 Cell lines representing early stages of CRC
Information obtained from the database search for cell lines relevant to study early stages of CRC can be found in Supplementary Table S3. There are several cell lines used to investigate CRC during all stages and the choice of a certain cell line is not always justified in literature studies. The cancer cells from which the cell lines originate are also harvested at different stages of the disease. Some are even taken from the metastatic site, and not from the colon itself. Commonly used cell lines include HT-29, Caco-2, SW480, SW620 and HCT116 (28). Cell lines SW480 and SW116 both have mutations on APC, KRAS and TP53. Cell lines SW1116 and SW480 are obtained from patients in stage I and stage II CRC, respectively (13). The cell line SW620 stems from CRC stage III and is derived from the same patient as cell line SW480 (13). The two cell lines SW480 and SW620 can therefore be used in comparative studies that focus on the disease progression.
3.3 Mouse models representing early stages of CRC
The mouse models APC-/+ and Fabpl-Cre;Apc15lox/þ are relevant to study early stages of
CRC (29,30). Further information on mouse models is listed in Supplementary Table S4. APC-/+ mouse model is used for early disease biomarker discovery (29). This is a commonly
used CRC mouse model, and it is heterozygote for APC. It develops about 30 small intestinal polyps that only occasionally progress to invasive adenomas (31). APC mice rarely develop metastases, and this has been helpful in studying genetic events during early stages of CRC (32). There are APC-/+ mouse models that have up to 300 polyps and most APC-/+ mouse die
young (4-5 months) (33). Fabpl-Cre;Apc15lox/þ is a mouse model that has an extended lifespan
compared to most APC-mutant models. Therefore, this model has a significant number of adenomas and adenocarcinomas in the large intestine. This makes this model more suited to study the adenoma-carcinoma sequence (30). Invasiveness and metastases are not reported (34). This mouse model is for example used by Fijneman et al. (35) to study protein biomarkers for early diagnosis of CRC.
3.4 Protein expression in human tissue samples from early CRC
In the search to find articles performing global proteomics analysis, those that were selected used human CRC tissues from early stages together with healthy intestine mucosa. A total of seven articles were found that fulfilled these criterions. The chosen articles are listed and summarized in Table 1. The original aim of these articles differ, but some of them investigate biomarkers in early CRC (16,36). Saleem et al. (37) investigated proteins associated with advancement of CRC, and the other articles investigated biomarkers in all stages (15,38–40). The methods used to investigate biomarkers also differ. Zhang et al. (38) and Hao et al. (15) they used LC-MS/MS. Sethi et al. (40) used label-free nano LC-MS/MS analysis, and Zhang et al. (39) used acetylation stable isotopic labeling coupled with L linear ion trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT MS). The remaining articles used iTRAQ labeling together with MS/MS (16,36,37). All these studies were performed in different laboratories. More information can also be found in
Supplementary Table S5.
Table 1. Overview of articles with proteomics analyses from early CRC human tissue samples identified and data processing to identify relevant biomarkers
Original data Results
Ref. CRC/healthy Tissue samples (#) Paired samples Yes/No CRC stages Proteins (#)a CRC/healthy tissue samples (#) CRC stages Proteins identified (#)b (36) 28/28 Y I-IV 10 452 15/15 I-II 11 (15) 22/22 Y I-IV 12 380 6/6 I-II 78 (40) 8/8 Y I-IV 948 4/4 I-II 10 (16) 21/21 Y I-II 4325 21/21 I-II 35 (38) 90/30 N I-IV 7526 50/30 I-II 255 (37) 8/4 N I-IV 2777 6/4 I-II 50 (39) 20/20 Y I-III 798 20/20 I-IIIc 17
a. Total number of proteins quantified.
b. Proteins identified that were significant upregulated (>1,5), p-value <0,05 and possibly localized in the plasma membrane.
c. Three samples out of 20 were from stage III.
In order to select relevant upregulated biomarkers, the data in the articles was further analyzed as described in section 2.4.Uzozie et al. (16) used q-value instead of p-values in their report. The q-value is a corrected p-value that takes into consideration multiple testing and the increased chance of error due to this. Q-values are not calculated in the same way as
p-values and therefore not comparable to the other studies, but nevertheless both give an indication of statistically relevant biomarkers.
From the identified, significantly upregulated proteins in early CRC, the ones located at the plasma membrane according to UniProt (18) were transferred to Microsoft ® Excel. This resulted in 383 different proteins (Supplementary Table S6). A plot diagram of the
significantly upregulated biomarkers reported with p-value can be seen in Figure 3a. A separate plot can be seen with the significant upregulated proteins reported with q-values in Figure 3b. Out of these, 61 proteins were found in at least two articles and were further investigated to find if they are expressed on the apical side of the intestinal epithelial cell membrane. To use results from at least two articles could strengthen the importance of the protein when different methods are used, and independent laboratories finds the same proteins.
Figure 3. Selected proteins are highlighted with different symbols and colors. a) Plot diagram showing –log P-values and log2 fold change for the upregulated proteins. The open circles represent those p-P-values where the value is only stated as <0,05 (but not specified further, those were set as p=0,05 in this graph). b) Plot diagram showing -log q-values and log 2-fold changes for the upregulated proteins.
3.5 Biomarker Selection
Further evaluations of the 61 proteins (see section 2.5) resulted in seven proteins that could be apically located in the colon during early stages of CRC. The seven selected proteins are Annexin A3 (ANXA3), B-cell receptor-associated protein 31 (BCAP31), Minor
histocompatibility antigen H13 (HM13), Solute carrier family 1 member 5 (SLC1A5), Solute carrier family 3 member 2 (SLC3A2), Solute carrier family 12 member 2 (SLC12A2) and Voltage-dependent anion-selective channel protein 1 (VDAC1). Table 2 contains information of the reported fold change in each article for the selected proteins. The table also contain
proteins identified in Figure 3. Immunohistochemically stained images of these proteins from human gastrointestinal tissue samples were found in the Human Protein Atlas (17) and give further indication about their apical membrane localization (Figure 4). Keep in mind these images originate from healthy colon tissues, hence the protein expression may differ in cancerous tissue.
Figure 4. Immunohistochemically stained colon tissue samples obtained from the Human protein atlas (17) for the seven selected proteins, illustrating their cellular localization.
Table 2. Selected proteins as apically expressed biomarkers for early-stage CRC. Protein expression during
CRC stage I-II Preclinical early CRC models
Protein expression during CRC stage I-IV Gene Fold changea P-valuea Ref. SW480b SW620c
Fabpl-Cre;Apc15lox/þ
Mouse modeld
Fold changee P-valuee Ref.
ANXA3 2,71 1,00E-08g (16) ✓ ✓ ✓i 2,37 9,00E-06 (15) 2,06 6,04E-14 (38) 2,06 1,30E-19 (38) 3,37 1,15E-02 (15)
BCAP31 2,10 3,46E-02 (15) 1,77 4,43E-05 (15)
4,72 <0,05h (39) 1,08 3,26E-01 (38) HM13 6,59 1,12E-20 (38) ✓ 1,38 2,73E-03 (15) 1,87 2,51E-02 (15) 6,10 6,78E-45 (38) SLC12A2 1,80 1,15E-06g (16) ✓ ✓ 3,27 3,45E-04 (15) 6,93 4,65E-02 (15) 1,59 <0,05h (37) 1,20 2,70E-02 (38) 3,16 <0,05h (39)
SLC1A5 1,81 3,24E-08 (38) ✓ ✓ 2,26 2,62E-06 (15)
5,12 2,62E-03 (15) 1,69 8,40E-09 (38)
SLC3A2 1,57 2,72E-05 (38) ✓ ✓ 2,47 1,74E-05 (15)
2,50 1,56E-02 (15) 1,49 1,18E-09 (38)
VDAC1 2,38 4,19E-02 (15) ✓ ✓ 1,56 5,59E-05 (15)
4,55 <0,05h (39) 1,13 6,00E-03 (38)
a. Fold change and p-value of early stages of CRC.
All proteins except SLC12A2 and ANXA3 were reported significantly upregulated in two different articles, whereas SLC12A2 in four and ANXA3 in three different articles. As seen in Table 2, four of the proteins were found to be less upregulated when including data from later stages of CRC (BCAP31, SLC1A5, SLC12A2 and VDAC1) and could thus be very interesting biomarkers specific for early stages of CRC. The proteins ANXA3, HM13 and SLC3A2 were upregulated to a similar extent when including later stages of CRC (Table 2). ANXA3, SLC1A5, SLC3A2, SLC12A2 and VDAC1 were all found in both cell lines SW480 and SW620. BCAP31 were found in none of these cell lines and HM13 only in SW480 (41). The only protein found in the mouse model was ANXA3, although the p-value is >0,05 (35).
4 Discussion
This work has resulted in the selection of 7 potential biomarkers that might be apically located and therefore available for targeting imaging probes from the luminal side of colon. The selected biomarkers might be of clinical importance as they are found in several studies using human tissue biopsies. The results need to be validated with further experimental studies regarding the proteins’ subcellular location. However, this work has provided a starting point for such future research and the development of orally administered in situ imaging probes.
If possible, more proteomic datasets could be investigated in order to have more supportive information to select biomarkers. Here, selection was first done on proteins that were reported in at least two articles, and with more datasets other proteins might have been found. There is a need for more datasets available for analysis where the data included are extensive, i.e. global proteomic analyses of human tissue samples from a diverse set of disease stages. For example, studies performed on human tissues when including further sample specific information would be helpful, i.e. patient data on sex, age, BMI, previous diseases like IBD etc., in order to link protein expression to certain patient populations. One disadvantage on this work is that the p-values and fold changes cannot be directly compared due to different methods in each study. It is mostly used to see whether the study itself has found a significant upregulated protein or not – but the number itself is not a directly comparable value across studies.
Search was conducted to find proteomics dataset for cell line SW1116 to be able to compare the found proteins and see if they also were expressed in this cell line. This cell line
was obtained from a patient in CRC stage I, i.e., the earliest phase of CRC. Unfortunately, the results were unsuccessful as no suitable dataset was found and instead the comparison was performed with cell lines SW480 and SW620.
The seven proteins that are reported upregulated during early stages of CRC and selected in this study are SLC12A2, SLC3A2, SLC1A5, ANXA3, BCAP31, VDAC1 and HM13. Three proteins are Solute Carrier (SLC) proteins that include over 400 transport proteins. SLCs transport substances across cell membranes. These proteins contain between 1 to 16 transmembrane domains and about 60 % of solute carrier proteins are annotated in the plasma membrane (42). Hellinen et al. (43) have separated the apical and basolateral
membranes of retinal pigment epithelium and analyzed the fractions to reveal the protein expression. All selected proteins except VDAC1 (not detected) were reported having a apical/basolateral ratio over >1,00 indicating they are apically located here (43).
ANXA3 is found to have about the same upregulated protein expression when including data from later stages of CRC compared to early stages (Table 2). ANXA3 facilitates tumor development and is indicated to promote adenoma to carcinoma progression (44). Usually, ANXA3 is reported to be in the cytosol and not in the plasma membrane. Annexins are involved in membrane transports and calcium-regulated activities on the cell membrane surface (45). However, in the immunohistochemical staining from the Human Protein Atlas (Figure 5), it seems to be in the plasma membrane. ANXA3 has been found to translocate to the plasma membrane in activated neutrophils and monocytes (44).
BCAP31 expression is decreased when including data from later stages of CRC compared to early stage (Table 2). BCAP31 has been reported as a cell surface candidate biomarker for adenoma-carcinoma progression (9). BCAP31 are a component of the plasma membrane and are exposed on the cell surface (46,47). Xu et al. (48) found BCAP31 to be upregulated during CRC stage I and II. Ma et al. (49) suggests that BCAP31 is a biomarker for CRC as it is upregulated in tumor tissue.
HM13 upregulated expression is around the same when including data from later stages as in early stages of CRC (Table 2). HM13 is a multi-pass transmembrane protein with its loop region on the extracellular side (50). It has also been reported as a cell surface candidate biomarker for adenoma-carcinoma progression (9).
SLC1A5 expression decreases when including data from later stages of CRC but is still upregulated compared to normal mucosa (Table 2). SLC1A5 is a plasma membrane
significant correlation between high SLC1A5 expression and KRAS mutation, that is relevant during early stages of CRC. The expression of SLC1A5 was high in 43.6% patients with mutated KRAS, compared to 24,1% for CRC patients with wild-type KRAS mutation. SLC1A5-knockdown results in reduced cell growth and increased cell apoptosis in KRAS-mutant CRC cells, it also results in the suppression of cell migration (52).
SLC3A2 expression when including data from later stages of CRC is about the same as in the early stages (Table 2). Sun et al. (53) found that there was an increase of SLC3A2 expression when CRC progressed from normal colon to carcinoma. Kucharzik et al. (54) reported SLC3A2 to be upregulated and epithelial expression enriched in mice during inflammation in the intestine. SLC3A2 is a transmembrane protein that has a large
extracellular domain (55). Is has been described that the protein redistributes to the apical cell surface during intestinal inflammation (56). This protein is also presented at the apical side of trophoblasts (57). Nanoparticles containing surface SLC3A2 antibody used for oral
administration have been tested to treat colitis in mice (58).
SLC12A2 is significant upregulated in four studies (vs two studies only for the rest of the proteins). The protein expression is reduced when including data from later stages of CRC, but still upregulated compared to normal mucosa (Table 2). This transmembrane protein has two substantial extracellular elements (59). SLC12A2 can be found on the apical side of choroid plexuses epithelial cells surrounded by cerebrospinal fluids in the brain (60). Merli et al. (61) suggests that SLC12A2 commonly is expressed in the lower crypts, but during onset of cancer stem cells it can be found in the upper portion of the crypts.
VDAC1 expression is decreased when including data from later stages of CRC compared to early stage (Table 2). VDAC1 is predominantly expressed in the outer
mitochondrial membrane as a porin ion channel. On the other hand it has also been reported to be expressed in the plasma membrane and are involved in regulating cell volume (62,63). VDAC1 has been found upregulated in CRC tissue compared to normal tissue (63).
5 Conclusion
This work has resulted in the selection of seven different proteins that are upregulated during early stages of CRC. The proteins are: ANXA3, BCAP31, HM13, SLC1A5, SLC3A2, SLC12A2 and VDAC1. These proteins are potentially located on the apical side of the intestinal epithelial cells and therefore possible targets for orally administered, monoclonal
antibodies. These findings might lead to a novel way for preventive patient screening and hopefully reduce the mortality for colorectal cancer.
6 Future outlook
The cell line SW116 would be ideal as an in vitro model to investigate early-stage CRC. Today, proteomic data is lacking to compare the protein expression in this cell line with human tissue samples. Therefore, global proteomics analysis of cell line SW116 should be conducted.
The proteins selected need further validation in order to determine their subcellular location. This can be done with immunohistochemical staining of cell lines SW1116 and SW480. If this successfully identifies these proteins to be apically located, further in vivo validation can be done to evaluate the possibility to target the selected proteins with monoclonal antibodies.
7
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