Genetics of Colorectal Cancer Racial Disparities

Abstract

This chapter describes genetics and epigenetics discoveries that have allowed investigators to better define cancer at the molecular level. Taking into consideration the expanse of the field of cancer, the focus will be on colon cancer as a platform to provide examples of techniques, recent discoveries, and translation of genetic studies to cancer care. In addition, this segment contributes to our understanding of racial and ethnic disparities in colon cancer and the use of -omic assessments as an application in cancer research. Thus, this section will provide an overarching view of cancer by defining the molecular characteristics of colon cancer; parameters of cancer disparities; and genetic factors that contribute to colon-tumor biology, specifically recent findings at the DNA, RNA, and protein levels. Importantly, the correlation of these factors with the immune system will be defined. This section ends with future directions for studying colon cancer in patients from medically underserved communities. In summary, this unit provides an introduction to how genetic and genomic investigations are helping to elucidate biological questions in an inclusive manner that will benefit patients on a global scale.

1. Introduction

According to the National Cancer Institute of The United States, cancer is defined as a disease in which cells grow uncontrollably and spread to other parts of the body [1]. Cancer can occur in any human tissue when cells lose control of cell division and multiply abnormally. Tumors, the accumulation of abnormal cells, can be limited to one location or invade into nearby tissues to form new tumors, a process known as metastasis. Cancerous tumors can be solid tumors, like colon cancer, or cancers of the blood, such as leukemias [1]. Regardless of the classification or tumor type, however; all cancers have common molecular features that have been identified as the hallmarks of cancer. These hallmarks encompass the biological abnormalities that are present in a cell to be classified as a cancer cell. They include the morphology, biology, metabolism, and genetic composition that are shared by all tumors.

The system used to organize the complexity of a cancer cell into simple hallmarks has been evolving for decades. Currently, there are 10 hallmarks of cancer. This includes self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion, and metastasis, reprogramming energy metabolism and evading immune response, genome instability and mutation, and tumor-promoting inflammation [2]. Collectively, these characteristics arise from not only studying the biology but also assessing the genetic and genomic processes of cancer cells. For example, many hallmarks are common to both benign (non-cancerous tumors) and malignant growths, such as the evasion of apoptosis or cell death and limitless replication potential. Thus, it is only through genetic and genomic studies that the identification of additional markers provides evidence that cancer cells share the presence of genetic mutations and genomic instability. This approach is called the mutation theory and it argues that carcinogenesis is a process that initiates with genetic mutations that allow for the hallmarks of cancer to develop in a cell lifespan [3]. Therefore, we will summarize genetic factors that contribute to the hallmarks of cancer.

First, we will address the selective growth and proliferative advantage of cancer cells. Normal cells depend on growth signaling of a strictly regulated cell cycle to proliferate and maintain tissue homeostasis. On the other hand, in cancer cells, the growth and proliferative signals are altered by mutations in genes that code for growth ligands, receptors, and other survival-signaling molecules involved in apoptosis [4]. Depending on the biological role that they play in proliferation, growth factors can be upregulated or downregulated. Increased levels may enhance tumor progression, whereas, lower than normal levels may result in the escape of the tumor from regulation. A well-studied example is that of the transforming growth factor-β (TGF-β), which can be an anti-growth ligand but has also been implicated in tumor progression through stimulating differentiation of cancer cells. The duality of genes like TGF-β can be the result of gene amplification, somatic mutations, or chromosomal translocations that may lead to fusion proteins and aberrant signaling [4]. A more complex example is the rat sarcoma virus (RAS) protein, which is active in 30% of all cancers. This protein is often altered as the result of missense mutations in its gene or inactivating mutations in one of its negative regulators, resulting in a variety of effects such as enhanced growth and proliferation, suppression of apoptosis, rewiring of metabolism, promoting angiogenesis, and immune evasion. Thus, mutations in the RASgenetic pathway can be implicated in multiple hallmarks of cancer [5].

Similarly, another key regulator of cell growth is the tumor protein 53 (TP53), which is the most often mutated cancer gene, altered in over 50% of sequenced tumors. The main function of TP53is to detect cellular abnormalities that include genotoxic stress, excessive signaling, nutrient deprivation, and hypoxia. In response to these triggers, TP53can stop cell proliferation, initiate DNA repair mechanisms, or activate terminal differentiation and apoptosis. Not surprisingly, the genetic alterations to this tumor suppressor gene are involved in virtually all hallmarks of cancer [6]. Therefore, mutations and genetic alterations are important contributors to abnormal cell signaling, proliferation, and inhibition of cell death-regulators.

In addition to unregulated growth and cell death, cancer cells can develop the potential to invade and proliferate outside their original tumor niche. Metastasis is the process that allows cancer cells to form secondary tumors and metastatic disease is responsible for over 90% of cancer-related deaths and involves several steps. For a cancer cell to become metastatic, it must invade through the extracellular matrix, promote angiogenesis and tumor vasculature, survive transport in circulation, and manipulate foreign microenvironments [7]. Most human carcinoma cells migrate collectively in an aberrant pattern. In the case of solid tumors, such as those seen in colon cancer, cancer evolves from epithelial cells that are normally immotile and tightly adherent to one another and the surrounding matrix. These cells acquire mobility by an epithelial-mesenchymal transition (EMT) that allows an epithelial cell to become mesenchymal [8]. The biological changes of EMT can include mutations on genes that are involved in epithelial growth factors, tissue hypoxia, metabolic and mechanical stress, and matrix composition. Mutations in EMT transcription factors could result in repressing epithelial genes and activating mesenchymal ones, or in epigenetic modifications that facilitate cancer cell invasion and migration. Once cells acquire the ability to invade new tissues, they adapt by proliferating in their new microenvironment.

One of the main strategies for cancer cells to thrive in new microenvironments and to promote cancer growth in their primary niche is to accumulate genetic and epigenetic modifications that are advantageous for metabolic rewiring. Metabolism in cancer cells may include changes in the use of glucose, amino acids, nitrogen, and alterations in metabolic gene regulation [9]. Among many nutrients, the most relevant ones are glucose and glutamine as they play a crucial role in carbon degradation, synthesis of macromolecules, ATP generation, nitrogen uptake, and nucleotide biosynthesis [10]. In the case of glucose, cancer cells have developed the “Warburg effect;” the increased utilization of glucose under aerobic conditions. This effect results from genetic and epigenetic changes that increase the transport and degradation of glucose, as well as in the deregulation of signaling pathways such as PI3K/Akt and the oncogenes KRAS proto-oncogene GTPase (KRAS) and the proto-oncogene serine/threonine kinase (BRAF). Although less studied, the increased demand of glutamine by cancer cells appears to be involved in the processes of protein synthesis, protein degradation under nutrient-deprivation conditions, engulfment and digestion of living cells, and phagocytosis of apoptotic products [11]. Cancer cells rely not only on genetic and epigenetic changes to promote metastasis but also on their interactions with the neighboring cells. Stromal cells such as fibroblasts contribute to the intratumoral cell heterogeneity, where cancer and normal cells will contribute to tumor growth and progression. Cancer-associated fibroblasts contribute to therapeutic resistance, the acquisition of nutrients, and evasion of the immune system [12].

Finally, a hallmark of all cancer types is the ability to evade immune surveillance. The immune system uses cancer-immunoediting to regulate and eliminate cells that proliferate uncontrollably. Immunoediting is made up of three phases: elimination, equilibrium, and escape. Elimination of cancer cells is the ultimate effect of the immune surveillance of the innate and adaptive immune systems. There must be a recognition of tumor cells by innate immune cells, a maturation and migration of antigen-presenting cells, the generation of tumor-antigen-specific T-lymphocytes, the activation of cytotoxic mechanisms, and, finally, elimination of tumor cells [13]. For cancer immunoediting to be efficient, the immune system should be able to generate the genetic and epigenetic changes that are required to interact with tumor cells that undergo antigen remodeling and selection. Cancer cells with reduced immunogenicity result in the production of resistant variants with mutations that increase resistance to immune cytotoxicity. These tumor variants are characterized by genetic and epigenetic alterations that reduce tumor-antigen recognition, increase resistance to cell death, and induce immunological tolerance [14]. Furthermore, and in correlation with other hallmarks of cancer, cancer cells can suppress the cytotoxic components of the immune system through the secretion of immunosuppressive factors and inflammation. For example, cancer and stromal cells can secrete proinflammatory molecules, tumor-derived exosomes can suppress the function of immune cells, and metabolic rewiring and altered microbiome can result in negative regulation of the immune system [14]. In summary, the immune modulation observed in tumors is currently recognized as a key player during cancer initiation and progression, and as a promising field for therapeutic manipulation.

Importantly, a major disparity in the prevalence, incidence and mortality of colorectal cancer (CRC) between African Americans (AA) and Caucasian Americans (CA) exists. Differences in response to treatment is an established factor influencing the overall survival of CRC patients. Here, we will address tumor biology and the importance of inclusivity in research. Knowledge of the molecular differences in CRC arising in different populations is needed to drive new therapeutic strategies and help overcome treatment resistance mechanisms to reduce disparities observed for this disease in minority populations. Depicted in Figure 1 is an overview of those factors associated with cancer initiation, progression, and health disparity. Although we focus in this report on tumor biology, it is important to note that social determinants of health factor strongly into health disparity. Therefore, it is imperative that we not only understand tumor biology but are also able to address social determinates of health with respect to overall survival of all colon cancer patients.

Figure 1.

Differences between AA and CA colon cancer patients at various levels of genomic expression and control; specifically, gene expression, DNA methylation, and chemotherapeutic response. Upstream factors are associated with and driven by social determinates of health. IND (investigational new drugs), SOC (standard of care), and NA (normal adjacent).

2. Types of cancer

As it was mentioned in the introduction, cancer can arise from any cell in the human body that grows in an uncontrollable manner [1]. When classified by the location, or tissue, of origin, there about 200 different kinds of cancers. Nevertheless, when we focus on the aberrant gene expression of cancer and how it relates to the cellular composition, it can be concluded that there are 5 types of cancer: carcinoma, sarcoma, leukemia, lymphoma and myeloma, and brain and spinal cord [1].

Carcinomas can develop from the epithelial cells from the skin or the tissue lining of internal organs. Therefore, they could be further classified depending on the “skin layer” where they are localized, namely as adenocarcinomas, basal cell carcinomas, squamous carcinomas and transitional cell carcinomas. Another type of cancer—sarcoma—arises from connective tissue such as bone, cartilage, adipose tissue, muscles and vascular tissue (i.e., blood vessels). Leukemia, on the other hand, is characterized by developing specifically in white blood cells and its primary source, the bone marrow. The lymphomas and myelomas, develop from cells from the immune system, from locations such as the lymph nodes and the spleen. Lastly, cancers from the central nervous system, will be present in the brain and the spinal cord [1].

Carcinomas are the most common type of cancer, with 85% of the cancer cases in the United States, and include lung and colon cancer, which are number one and third in terms of incidence, respectively [1]. Sarcomas on the other hand, compose less than 1% of the cancer cases and most of the diagnosis are bone sarcomas [1]. Although leukemias are only the 3% of cancer cases in the United States, they are the most common type of cancer in children [1]. Cancers of the lymphatic system—lymphomas and myelomas—are 5% and 3% of the cancer cases, respectively. These cancers are particularly challenging to treat as they arise from abnormalities in the bone marrow, and they commonly require bone marrow transplantation [1]. Finally, brain and spinal tumors are 3% of the cancer cases in the USA and the most frequent subtype is the brain tumor from glial cells [1]. Taken together, it can be concluded that there are several cancer types and subtypes, based on the cellular composition of the cancer source. It is important to be noted, however, that many cancer types share aberrant gene expression that can cross over cancer types and subtypes. Therefore, we will address the shared genetic transgressions across the most common cancer types.

3. Aberrant gene expression in cancer

To address the observed abnormalities of gene expression across cancer types, we will start by describing the molecular basis for cancer progression. Mutations that lead to oncogene (stimulators of cell division) activation and tumor suppressor (regulators of cell cycle) dysregulation influence cancer initiation and progression. Such aberrant gene expression will, in turn, contribute to the hallmarks of cancer: unregulated growth and cell death. It is important to emphasize that the functional distinctions of mutations on the same gene from one cancer classification to another relies on the cellular differences and the distinct pathways that are deregulated in each cancer type.

Not surprisingly, most oncogenes and tumor suppressor genes are components of pathways that are involved in cell signaling. They are responsible for the regulation and generation of molecular signals, such as proteins, receptors, ligands, etc. For example, mutations in receptors of the tyrosine kinases RAS family, may act as oncogenes that are present in up to 80% of carcinomas [4]. Other signaling molecules that normally act as tumor suppressors, such as the TGFβ family, can lose regulatory function and commonly present as constitutively active receptors due to mutation [4]. An important example of aberrant gene expression in key pathways of cellular proliferation are those that are involved in DNA repair and cell division. A clear point of reference is the tumor suppressor gene RB. Mutations in the RBgene result in defects in the RB protein that normally acts as a restrictive molecule for cells to enter the S phase in the cell division cycle, leading to cancer cells proliferating inappropriately [4]. Interestingly, aberrant gene expression of the RBgene of the RB-regulatory pathway, including cyclins and cyclin-dependent kinases, are shared by many cancer types. The alterations of this pathway can be found in drastically different types of cancer: brain cancers (i.e., glioblastomas and carcinomas) and breast cancer, for example. Such observations highlight the importance of understanding gene pathways and their mechanism of action at the molecular and cellular level for improved targeted clinical outcomes.

The extent of the contribution of a single gene to the development of different cancer types is best exemplified by the aberrant mutations in the TP53gene [6]. As it was mentioned in the introduction, mutations in the TP53gene allow cells to survive and proliferate despite DNA damage. This tumor suppressor gene is present in 85% of human cancers and it is arguably the most important gene in human cancer, regardless of the cancer type. Hence, aberrant gene expression of TP53can simultaneously dysregulate the cell cycle, apoptotic signaling, and the overall genetic stability of a cell. The cascade reactions that are produced by abnormalities in the TP53pathway include alterations in the p21 pathway, the CDK complex, the MMR DNA repair system, among many others, making this gene relevant in virtually any cancer type [6].

Taken together the hallmarks of cancer and overlapping aberrant alterations in gene expression across cancer types, it is appropriate to conclude that using a type of cancer as a model of study, could allow us to better understand the genetics of cancer as a whole. Thus, we have selected colon cancer to illustrate some of the general principles and molecular mechanisms of tumor progression due to aberrant gene expression. Considering the global prevalence of colon cancer, responsible of about 11% of the cancer deaths, along with its defined stepwise genetic hallmark timeline, this cancer type seems to be the perfect prototype to address cancer gene expression. In addition, colon cancer provides researchers with the opportunity to study the physical progression of a tumor in the human epithelium along with the molecular changes that result from aberrations at the gene expression level.

4. Genetic factors that contribute to colon cancer

Colorectal cancer (CRC) is one of the most common cancers across the globe. It is estimated that the incidence of colon cancer will increase by 60% in several countries, positioning CRC as the second deadliest cancer type [15]. Up to 90% of CRC tumors arise from adenocarcinomas from the colon and rectum and up to 65% of the cases are sporadic (without a family history of CRC) [16]. Thus, the majority of CRC tumors progress from somatic and epigenetic alterations from modifiable risk factors such as metabolic comorbidities (e.g., obesity), diet, smoking, and alcohol consumption, among others [16]. It is important to highlight, however, that regardless of their inherited or somatic nature, several genetic factors and pathways have been identified in the pathogenesis of CRC.

In cases of hereditary CRC tumors, approximately 5% are classified as familial adenomatous polyposis (FAP) induced by alterations in the adenomatous polyposis coli (APC), MutL homolog 1 (MLH1), or the MutS homolog (MSH2) genes [17]. These inherited genetic factors are the result of chromosomal instability (loss or gain of chromosomal segments), aberrant methylation (altered CpG islands), and (or) microsatellite instability (MSI) due to the loss of DNA repair machinery [18]. These genetic alterations can also arise from somatic mutations (non-hereditary) of tumor-associated genes, for instance cell cycle regulators, which will in turn contribute to the aforementioned hallmarks, tumor initiation and progression [18].

Hence, either from hereditary factors or from genetic changes during the life span of the patient, CRC is determined by several genetic pathways. Although CRC tumors are considered heterogeneous at the molecular level, all tumors from the chromosomal instable pathway (CIN) have in common the accumulation of mutations after cell division cycles and loss of chromosomal stability [19]. This pathway is characterized by increased mutation rates, alterations in chromosome number, and rearrangement of chromosomes, which are detected by karyotyping and DNA analysis [19]. Some of the mechanisms by which CIN contributes to CRC tumorigenesis are mutations in key, cell cycle-related genes. These key genes include BRAF, KRAS, TP53, and importantly, the tumor-suppressor APCgene which is responsible for familial adenomatous polyposis and 85% of colorectal cancer cases without a hereditary risk factor [20]. In short, the tumor progression of the CIN pathway encompasses the few steps from polyps (adenomas), that can turn normal colorectal epithelium into solid tumors (adenocarcinomas): mutations on the APCgene in epithelial cells, mutations in the KRASgene in adenomas, inactivation of the tumor-suppressor gene TP53on chromosome 17p, and the deletion of chromosome 18q [20]. Other genes that are closely associated with the functioning of the APCgene are also altered in CRC. One example is the CTNNB1(β-catenin) as mutations in the APCgene result in the overproduction of β-catenin in the cytoplasm and the overactivation of the Wnt signaling pathway [20]. Likewise, mutations in the KRASgene will upregulate the activation of mitogenic pathways such as the mitogen-activated protein kinase (MAPK) pathway, the deleted in the colon cancer (DCC) pathway, and the TGF-β signaling pathway, to name examples [21]. Lastly, mutations in the TP53gene are present in 43.28% of CRC cases (mostly missense mutations); they impair the cell’s ability to respond to stress, to execute DNA repair, and to arrest cell cycle or implement apoptosis [21].

Following CIN, the second most relevant pathway in CRC is microsatellite instability (MSI). These tumors are characterized by genetic damage of the mismatch repair (MMR) system and they usually present inhibition of the DNA polymerase, that in turn creates short-term insertion-deletion loops (IDL) that further enhance genetic instability [22]. MSI tumors are clinically identified by the abnormal production of the proteins of the DNA MMR system: MLH1, MSH2, MSH6, and PMS2; whose main functions are to repair single base pair mismatches during DNA synthesis and to maintain genomic stability after each cell replication cycle [22]. MSI mostly occurs in the proximal colon and its classification is used as a biomarker for prognosis and standard of care (immunotherapies) based on the 5 microsatellite markers: mononucleotides BAT25 and BAT26, and the dinucleotides D2S123, D5S346, and D17S250 [23]. Moreover, CRC tumors can be classified as MSI-high (MSI-H), those with >30% markers that exhibit genetic instability, MSI-low (MSI-L) for tumors with less than 30% markers with instability or, microsatellite stable (MSS) for tumors with no genetic stability and normal production of the MMR proteins [23]. Contrary to the CIN pathway, MSI tumors are for the most part somatic defects in the MMR genes by either mutational inactivation or by epigenetic silencing of CpG due to aberrant methylation patterns that result in the silencing of promoter genes, although hereditary mutations are frequently reported in MSH2, MSH6and PMS2. In conclusion, MSI pathogenesis arises from the accumulation of mutations and/or epigenetic alteration in several genes rather than the initiation from a single driver [23].

To finalize our genetic factors in the CRC section, we will address the CpG island methylator phenotype (CIMP) serrated pathway. Tumors classified as CIMP have a high number of hyper-methylated genes, with promoters that are silenced and can cause the downregulation of gene expression and protein production [23]. Approximately 35% of CRC cases are CIMP and they have common molecular alterations in other pathways such as the MSI subtype, the hypermethylation of the MLH1gene (part of the MMR system) that can lead to DNA repair dysfunction, and the development of hyperplastic polyposis syndrome that also involves the APCgene and it is commonly associated with hereditary Lynch syndrome [23]. Overall, CIMP and the serrated CRC tumor subtypes develop faster than other somatic pathways as they combine the rapid accumulation of polyps followed by accumulation of mutations from the defective MMR system and the further silencing of cell cycle genes. Taking all these pathways together, it can be stated that genetic factors in CRC can be hereditary and somatic, with overlapping mutations in key cell cycle genes that contribute to the adenomatous-to-adenocarcinoma transformation in the colon epithelium.

5. Tumor biology composition in African American colon cancer patients

To address the specifics of the tumor biology of CRC among African American patients, we would like to start by describing the concept of cancer disparities that are observed in the United States of America (USA). The National Institutes of Health considers the main racial/ethnic minority groups to be African Americans, American Indians and Alaska Natives, Native Hawaiians and Pacific Islanders, and Hispanic/Latinos [24]. These minority populations are heterogeneous, and we acknowledge that these categories are socially constructed; however, they serve as the official method for tracking cancer incidence, progression, outcomes, and all the other metrics by which cancer care and research is organized in the USA. Hence, this section will start by providing a summary of the cancer disparities that are observed among underrepresented CRC patients and it will continue by focusing on the biological factors that have been studied in African American patients.

There are disparities in the incidence and mortality rates of CRC among all the mentioned minority groups when compared to the average USA population, which is in the majority composed of Caucasian Americans (CA) [24]. For instance, in terms of incidence, it is 45.7 for African American (AA) patients, and 34.1 for Latinos per 100.000 patients. Similar trends are reported for mortality rates, with 19.0 for AA and 11.1 for Latinos, indicating that although progress has been made in the prevention and treatment of CRC in the USA, these patients face challenges that are population-specific [24]. Many factors contribute to this phenomenon, including socioeconomic status and access to healthcare, among others; nevertheless, there are unique biological features that contribute to the tumor biology of CRC in each racial/ethnic group. In the case of AA and Latinos, it is worth mentioning the disparities in the risk of metastasis due to the reduced prevalence of tumors that are localized and regional (normally cured by surgery or radiation) when compared to CA patients, underscoring the biological differences between these populations [25].

As we discussed previously, CRC results from a combination of the patients’ genetic profile (hereditary factors) as well as environmental and somatic changes that will modulate the tumor biology of the colon, such as diet, body mass index, tobacco and alcohol intake, etc. In addition to these contributors, research has highlighted tumor biology that has been associated with each population and their influence in the response to treatment. Studies from clinical trials have shown racial/ethnic disparities in survival rates of stage III CRC cancer even when patients received the same standard of care, greater toxicity in response to 5-fluorouracil (5-FU) therapy regimens, and unique pharmacogenetic variants in AA patients [26]. Also, the frequency of the MSI/MMR-deficient tumor subset, which is associated with better prognosis and serves as a biomarker for immunotherapies, appears to be reduced in AA when compared to CA patients (14 in CA vs. 7% in AA) [27].

Regarding the inherited, germline-associated syndromes in CRC, such as the familial adenomatous polyposis, AA seem to have a prevalence comparable to the other populations in the USA [28]. Despite that, previous investigations have identified unique somatic mutations in the 5-Hydroxytryptamine Receptor 1F (HTR1F), Folliculin (FLCN), and EPH Receptor A6 (EPHA6) protein-genes that in AA colon cancer patients seem to play a role in worse prognosis and greater chemoresistance [29]. These novel somatic mutations in AA suggest that EPHA6and FLCNcould serve as driver genes for CRC in these patients and they pinpoint the need for genetic studies that target specific populations. One of these studies demonstrated that the secretion of the interleukin-6 cytokine and oxidative species in the colon epithelium could impair the functioning of the MSH3 protein (MMR system) and promote the development of MSI-L, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). EMAST is a subtype that is more prevalent in AA patients when compared to CA [30]. These findings, in correlation with the reported lower rates of MSI tumors among AA patients, may encourage researchers to further investigate the influence of inflammation and the intersection of the immune system with the tumor biology of CRC in AA patients.

6. Role of miRNAs in colon cancer and African Americans

microRNAs (miRNAs), powerful regulatory RNAs 18–24 nt in length, play a major role in oncology pathways. As miRNAs can potentially serve as biomarkers for CRC at several levels, understanding miRNA dysregulation is important in defining its effects on biomolecular pathways and developing possible therapeutic targets. This section will discuss miRNA expression patterns characteristic of CRC tumors, miRNAs identified as potential non-invasive biomarkers, their role in chemo-response, their contribution to racial health disparities, and their use as therapeutic targets.

miRNA dysregulation is a common component of many cancers, including in CRC. Among the hundreds of discovered miRNAs, the most relevant miRNAs unique to CRC tumors compared to healthy tissue, as well as their primary oncological function, is summarized in Table 1 (upregulated miRNAs) and Table 2 (downregulated miRNAs). Notably, available data from The Cancer Genome Atlas was used to provide a comparative analysis of miRNA expressed in colon tumors (N = 253) versus uninvolved normal tissues (N = 8). Of these, 39 upregulated and 54 downregulated miRNAs were implicated in colon cancer and, 9 of them were critical with a downstream impact on 461 genes associated with patient survival [85]. Among others, pathways affected by these miRNAs include Wnt signaling, p53, cell adhesion, cAMP signaling, stem cell pluripotency, MAPK, and HIF-1 [85]. In the pathway network of these miRNAs, five ‘hub’ genes (genes that have high connectivity to oncological pathways) were identified as mediating the function of these miRNAs: PPARGC1A, COLIA1, SYT1, PGR, and KCNB1[85]. Additionally, a study of patients with stage III CRC showed that 11 miRNAs (miR-135b, miR-141, miR-18a, miR-20a, miR-21, miR-224, miR-29a, miR-31, miR-34a, miR-92a, and miR-96) were overexpressed in tumors relative to their matching normal samples [86]. In addition to deletion and mutation, hypermethylation of certain miRNA promoters contributes to the increased dysregulation of miRNAs in CRC [87].

[CRC tumor miRNA profile] summary of upregulated miRNAs (tumor vs. normal tissue)
Biological function	Upregulated miRNAs	Reference
Promotes cell cycle/proliferation	17-3p, 20a, 21, 26a, 31, 106a, 135a/b, 141, 200c, 301a, 598, 1273g-3p	[31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42]
Promotes migration/invasion/metastasis	20a, 21, 26a, 29a, 31, 135a/b, 155, 200c, 224, 301a, 494, 1273g-30	[32, 33, 34, 35, 37, 39, 40, 42, 43, 44, 45, 46]
Inhibits apoptosis	17-3p, 31, 92a, 106a, 135a/b, 200c	[31, 35, 36, 37, 39, 47]
Involved in drug sensitivity/resistance	96, 155, 192/215	[44, 48, 49]
Hypoxia/ROS regulation	210	[50]
Malignant transformation	182/503	[51]
CRC stem cell tumorigenicity	221	[52]
Ambiguous	18a (both an oncomiR and Tumor Suppressor), 217 (inhibits proliferation/promotes apoptosis)	[53, 54]

Table 1.

Upregulated miRNAs in CRC tumor tissue vs. normal tissue.

[CRC tumor miRNA profile] summary of downregulated miRNAs (tumor vs. normal tissue)
Biological function	Downregulated miRNAs	Reference
Inhibits cell cycle/proliferation	7, 18a-3p, 27b, 30a, 101, 125a/b, 126, 143/145, 144, 149, 155, 186-5p, 194, 205-5p, 216a-3p, 320a, 330, 374b, 375, 383, 455, 486, 511, 744, 1271, let-7	[53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79]
Inhibits migration/invasion/metastasis	7, 19b-1, 26b, 27b, 101, 125a/b, 126, 155, 186-5p, 205-5p, 320a, 328, 330, 374b, 455, 511, 744, 1271	[55, 56, 58, 59, 60, 64, 65, 67, 69, 70, 71, 74, 76, 77, 78, 80, 81, 82]
Promotes apoptosis	7, 18a-3p, 30a, 101, 129, 143/145, 149, 155, 455, 511, 744, 1271, let-7	[53, 55, 57, 59, 63, 64, 74, 76, 77, 78, 79, 83]
Involved in drug sensitivity/resistance	26b, 328, 1271	[78, 81, 82, 84]
Involved in angiogenesis	375	[72]

Table 2.

Downregulated miRNAs in CRC tumor tissue vs. normal tissue.

The feasibility of using miRNAs as biomarkers stems from their general stability in blood, owing to their structure which resists RNAse-mediated degradation [88]. miRNAs that may serve as CRC non-invasive biomarkers are summarized in Table 3 [101]. Thus, exosomes-circulating miRNAs may have value as early, non-invasive CRC biomarkers [89]. For example, serum from CRC patients contained 69 miRNAs that were significantly upregulated compared to normal subjects. Additionally, cell culture media from five CRC cell lines demonstrated 52 upregulated miRNAs compared to culture media from normal colon epithelial cells. For both these in vivoand in vitrodata, 16 miRNA (let-7a, miR-1224-5p, miR-1229, miR-1246, miR-1268, miR-1290, miR-1308, miR-150, miR-181b, miR-181d, miR-1915, miR-21, miR-223, miR-23a, miR-483-5p, and miR-638) were commonly upregulated compared to a non-cancerous baseline. Following surgical resection of tumors in 29 CRC patients of all stages (I-IV), serum levels of eight biomarker miRNAs (let-7a, miR-1229, -1246, -1224-5p, -150, -21, -223, -23a) were significantly decreased compared to pre-resection levels [89]. Importantly, significantly higher serum levels of miR-18a and miR-29a were observed in CRC patients when compared to levels in healthy individuals as control [86]. Therefore, circulating miRNAs have emerged as potential non-invasive predictive biomarkers for CRC.

[Diagnostic biomarkers] summary of upregulated in serum/plasma (CRC vs. healthy patient)
Location of biomarker	miRNA	Reference
Upregulated in serum (CRC patient vs. healthy control) ANDculture media (CRC cell lines, n = 5, vs. colon epithelial cells)	21, 23a, 150, 181b/d, 223, 483-5p, 638, 1224-5p, 1229, 1246, 1268, 1290, 1308, 1915, let-7A	[89]
Upregulated in serum (CRC patients vs. healthy control)	18a, 24, 24-2, 29a, 122, 135a-5p, 139-3p, 139-5p, 203, 320a, 423-5p, 6826	[86, 90, 91, 92, 93, 94, 95, 96, 97]
Markers in plasma exosomes	17-5p, 21, 92a-3p, 6803-5p	[98, 99, 100]

Table 3.

Summary of miRNAs that have potential as non-invasive biomarkers.

Since miRNAs modulate drug targets directly or indirectly, response to standard of care (SOC) chemotherapeutic agents may be influenced by the dysregulation of select miRNAs. Indeed, expression of miRNAs is altered upon treatment of CRC cell lines with 5-fluorouracil (5-FU). Patients who did not respond well to fluoropyrimidine chemotherapy had higher plasma levels of miR-106, miR-484, and miR-130b [102]. Furthermore, higher miR-27B, miR-148A, and miR-326 levels were associated with decreased progression-free survival, whereas miR-326 was related to decreased overall survival [102]. Additionally, 5-FU reduces miR-200b, which lowers the levels of the protein tyrosine phosphatase, PTPN12, which, in turn, downregulates oncogenes, including c-ABLand RAS, resulting in decreased cell proliferation [103]. Further assessment of these miRNAs in drug uptake and metabolism will help characterize their significance in chemotherapeutic response and pave the way for more personalized treatment plans.

Lacking in our understanding, due to the absence of or limited use of AA samples in bench and clinical studies, is the role of miRNA in CRC racial health disparity in terms of cancer initiation and chemo-response. Microarray and qPCR analysis implicated miR-182, -152, -204, -222, and -202 when comparing AA and CA tumor samples [104]. Among these, miR-182 was the most significant—upregulated in AA vs. CA—in race tumor interactions. FOXO1and FOXO3A, miR-182 targets, were shown to be downregulated in AA colon tumors compared to the colon tumors of CAs [104]. Findings by Bovell and colleagues “suggest that the prognostic value of miRNAs in colorectal cancers varies with patient race/ethnicity and stage of disease.” [105]. 5 miRNAs (miR-20a, -21, -106a, -181b, and -203) in paired normal and tumor CRC had higher expression in CRC than in adjacent non-involved tissues. High expression of miR-203was associated with poor survival of CA patients with stage IV CRC and with poor survival of AA patients with stages I and II colorectal cancers. High expression of miR-21and miR-181bcorrelated with poor survival of CA (stage IV) and AA (stage III) patients, respectively. These analyses suggest that a deeper biomolecular understanding of miRNA dysregulation between racial and ethnic groups may provide a richer context in addressing the clinically observed health disparity through more personalized treatments.

The ability of miRNA to regulate expression of many downstream genes and the proficiency of biotechnology to synthesize oligonucleotides, promote and enable the use of these molecules as potential therapeutic agents. miR-34a, a central miRNA in the p53 stress pathway, is often lost in CRC and has been the hallmark of miRNA mimic therapy. In a phase 1 clinical trial, liposomal miR-34a mimics were shown to provide benefits against advanced solid tumors; however, these mimics were accompanied by many off-target-side-effects and adverse immune reactions [106]. Other strategies may include inhibiting oncogenic miRNA or more specific targeting of tumor miRNA replacement therapy. The challenge of miRNAs as therapeutic agents is a limited understanding of their targeted pathways in various tissues.

miRNAs have become vital as prognostic and therapeutic biomarkers/targets for cancer. miRNA dysregulation in CRC, along with their role in racial health disparities, is continually being explored. Identification and profiling of miRNAs in diverse patient population will result in the generation of personalized therapeutic targets which will allow for optimal patient care.

7. Methylation patterns and alterations in colon cancer in African Americans

A major component of gene expression is DNA methylation. It is well documented that aberrant methylation patterns in CRC contribute to tumorigenesis and progression. This section will discuss dysregulated methylation patterns of CRC; specifically, pathways affected by aberrant methylation (hypo−/hypermethylation) and differential methylation of CRC tumors of African American patients.

In general, 10–40% of CRC tumor cells have a hypomethylated genome [107]. Most of this hypomethylation occurs in repetitive elements and influences the initiation of tumorigenesis [108]. In vivo, mice possessing a knockout of DNA methyltransferase displayed increased genomic instability and tumor initiation [109]. While methylation can alter gene promoters, methylation of non-coding regions like long interspersed nuclear elements (LINE) can also affect adjacent gene expression. In addition, hypomethylation of regulatory elements may lead to unregulated oncogene expression [110]. It is important to mention, however, that methylation of a gene/gene promoter is not necessarily a guarantee of decreased gene mRNA expression in the cell and that more complex downstream mechanisms may be at play [111].

In addition to hypomethylation, specific promoters involved in CRC can be hypermethylated. While hypermethylation of tumor suppressors is a normal part of aging, some methylation patterns describe preferential hypermethylation of tumor suppressors [112]. For example, the CpG-island methylator phenotype (CIMP) produces a subtype of CRC in which CpG islands of tumor suppressor genes become hypermethylated through an epigenetic instability pathway [112]. As proposed by Ehrlich and colleagues, the consequences of aberrant methylation include genomic instability, epigenetic inactivation of tumor suppressors, altered chromatin heterostructure interactions, and activation of oncogenic elements [107].

Specific CRC-relevant pathways are affected by methylation. A few are described here. In Wnt signaling, the APCpromoter is methylated in about 18% of CRCs. However, this does not necessarily correlate with a subsequent decrease in APCexpression of downstream target expression [113]. Furthermore, methylation of Wnt inhibitors (SFRP1, 2, 4, and 5) is an early event common in CRC [114]. Wnt signaling malfunction is an early hallmark and driver of CRC progression [115]. Therefore, if intrinsic inhibitors of Wnt signaling, like SFRPs, are downregulated via methylation, the cell may have a propensity for aberrant upregulated Wnt signaling [114]. In the p53 pathway, while direct methylation of TP53is rarely observed, components like p14-ARF (which sequesters MDM2 ubiquitin ligase) have been methylated and downregulated in 20% of microsatellite instable (MSI) CRCs [116]. For the RAS pathway, up to 80% of CRCs have RASSF1/2promoters methylated. These proapoptotic gene products are modulators of the RAS pathway. Thus, RASSF1/2 downregulation may promote tumorigenesis [117]. Finally, TSP-1—an extracellular matrix glycoprotein—cleaves TFGβ into the active form. This gene is often methylated at its gene promoter in about 20% of CRCs [118].

Aberrant methylation in CRC has been shown to stem from several sources. First, deregulation of relevant methylation enzymes like DNA methyltransferases (DMNT) can kickstart aberrant methylation. While DMNT is rarely mutated in CRC (unlike other cancers), the protein is overexpressed but not related to a specific aberrant methylation phenotype [119, 120]. TET1methylation has been suggested in the progression of CIMP CRC [121]. Mechanisms that normally protect the genome from aberrant methylation (e.g., DNA-binding proteins, RNA polymerase, or histone binding) may be modified which allows nearby hypermethylated regions to affect previously nonmethylated areas [122]. Specifically, Turker proposes a hypothesis wherein long-term methylation of nearby promoters initiates due to a constantly shifting methylation ‘boundary’ of adjacent hypermethylated regions [122]. As an unmethylated gene promoter couples and decouples with transcription factors, the ‘boundary’ of the adjacent hypermethylated region is in flux and can thus spontaneously/iteratively spread towards nearby CpG islands in the gene promoter, ultimately favoring a gene-repressing DNA superstructure [122]. Additionally, in response to oxidative stress, the DNA damage repair system recruits DMNTs which are implicated in methylation of nearby promoters [123]. Currently, DNMT inhibitors are being examined for use as an adjunct therapy to canonical chemotherapeutics for CRC; specifically, for prevention of the hypermethylation of tumor suppressor genes. These therapies have had some success in vitroand in mouse models, but not in clinical trials [112].

Importantly, it was reported in one study that the CRC tumors of AA patients had 14.6-fold more hypermethylated regions and 25-fold more hypomethylated regions than CA in respect to tumor versus normal tissue [124]. In AA tumors, CHL1, four inflammatory genes (NELL1, GDF1, ARHGEF4, and ITGA4), and 7 miRNAs were methylated. Of these miRNAs, miR-9-3P and miR-124-3P are implicated in CRC while the targets of miR-124 (which was hypermethylated) were upregulated in AA vs. CA [124]. In a separate study, four methylation target genes were observed in AA samples: BPM3, EID3, GAS7, and GPR75[110]. Differential methylation patterns in different groups of patients may inform more efficacious personalized treatment protocols, particularly in the field of DMNT inhibitors.

In addition to mutations, dysregulation of gene expression through epigenetic alterations (i.e., methylation) impacts the initiation and progression of CRC. However, while global hypomethylation and local hypermethylation are prevalent, more specific patterns may need to be analyzed for therapeutic consideration. Beyond the comparisons of CRC tumors to normal tissue concerning methylation patterns, although limited in the scope of race and ethnicity, there are also marked differences between tumors originating from different racial groups. However, these findings may be instrumental in predicting tumor aggressiveness and responsiveness to standard of care and novel treatment modalities.

8. Immunological profiles of colon cancer in African Americans

As we discussed in the introduction, evasion of immune surveillance is one of the hallmarks of cancer cells. Hence, we will discuss how genetic factors, tumor biology, and genetic regulators (miRNAs and DNA methylation patterns) intersect with the immune system in CRC in AA patients.

Colon tumors closely interact with immune cells that reside at the tumor site (microenvironment) and immune cells that are part of the systemic immune surveillance system. They both play a role in tumor progression, prognosis, and response to treatment in CRC [125]. Lymphocytes, cytotoxic CD8⁺ T cells to be precise, are one of the most efficient immune cells to perform surveillance, to limit the tumor progression in CRC and their filtration into tumors and are associated with better prognosis and outcome. The presence of high levels of CD8⁺ T cells in the center and invasive margins of colon tumors have been associated with improved patients’ survival when compared with patients with low infiltration of the same cell type [126]. These lymphocytes can ignite apoptosis in target cells by the secretion of (among others) the serine protease Granzyme B. Granzyme B⁺ T and Natural Killer (NK) cells are activated in response to the presence of neoantigens in the surface of cancer cells (recognized by antigen-presenting cells); especially from hypermutated tumors that are mostly classified as MSI due to their genetic instability and MMR deficiency [126]. In the context of AA CRC patients, however, it has been demonstrated that in MSI-H tumors, these patients presented lower infiltration of CD8⁺ T cells when compared to tumors from the same subtype from CA patients [127]. Furthermore, a study that investigated 250 CRC cases and compared MSS tumors from AA and CA patients found that tumors from AA patients had lower numbers of GRANZYME B⁺ lymphocytes, suggesting that CRC in AA is characterized by impaired immune surveillance and lower cytotoxicity regardless of tumor type [128].

These disparities in the immunological profile of CRC tumors in AA patients also influence the access of these patients to the immunotherapies available for CRC cancer. For example, when cytotoxic T cells are activated by tumoral antigens they will induce memory T cells that are characterized by the expression of the programmed cell death protein 1 (PD-1) receptor on their surface that serves as a negative feedback loop for the inactivation of these lymphocytes and prevents auto-immunity [129]. This receptor will interact with the PD-1 ligand that can be on the surface of cancer cells as a tumoral strategy of immune-surveillance evasion, and it is the target of the PD-1/PD-L1 antibody therapy that blocks this interaction and releases CD8⁺ T cells from the negative effects of the PD-L1 ligand. Not surprisingly, and as we mentioned in the tumor biology section, the MSI tumor classification is a biomarker for access to this immunotherapy based on the direct correlation of hypermutation in cancer cells, expression of neo-antigens and PD-L1 ligands, response to the anti-PD-1 antibody therapy, and positive outcome [130].

Remarkably, a research study that compared the gene expression of CRC tumors from AA and CA patients confirmed that tumors from AA had a lower expression of the GZMBgene (which codes for the GRANZYME B protein) and lower expression of PDL1(gene encoding for the PDL1 ligand), results that correlate the previously described findings at the protein and cellular levels [131]. Furthermore, this investigation demonstrated that colon tumors from AA presented significantly higher numbers of exhausted (or functionally impaired) CD8⁺ T cells and instead, had higher numbers of pro-inflammatory myeloid cells that are associated with chronic inflammation and worse prognosis. Lastly, the authors measured the levels of T cell-related cytokines in the plasma from both cohorts and demonstrated that CA had significantly higher levels of interleukin 12 and other CD8⁺ T cells-activating cytokines when compared to AA, proposing that the immunological profile of these patients present disparities at the tumor site and at the systemic level [131]. The combined conclusions from these studies in AA patients demonstrated that there is a lower incidence of MSI-H tumors in this population, lower infiltrations of cytotoxic T cells in MSI-H and MSS tumor subtypes, and a reduced gene expression and activation of GRANZYME B⁺ cells. Taken together, these research studies indicate that AA patients present an impaired immunosurveillance mechanism and may have lower access to the PD-1/PD-L1immunotherapy for CRC cancer.

9. Future directions of gene expression and colon cancer in African Americans

The push for research in CRC racial health disparity and inclusivity in clinical trials represents a major step forward in personalized medicine and optimizing health outcomes for a diverse patient population. To continue the acceleration of current progress, new future directions in the analysis and development of novel in vitroand in vivomodels are necessary. In terms of analytical frameworks, multi-omics assessment of CRC will help unlock a new level of comprehension and possible treatment pathways. These types of analyses, often including genomic, epigenetic, and expression analyses, can reveal a richer set of conclusions and holistic understanding of the disease and underlying racial health disparity [132]. This perspective can be elaborated when considering synergy with other physiological analyses. Intersectionality between gene expression and metabolomics studies can supply a detailed description of compound effects and mechanisms. For example, studies with the compound shikonin were analyzed with CRC through integrated transcriptome and metabolomic perspectives which can allow for more robust predictive conclusions [133]. Mouse model studies demonstrate the ability of microbiome dysbiosis to epigenetically influence gene expression profiles of the colonic epithelium towards more inflammatory or CRC risk patterns [134].

Not only will these multidimensional analyses aid the progress of CRC research, but so will the development of tools that seek to make racial health disparity research more accessible. Social determinants of health are important components of health disparity; however, collective studies have demonstrated that research observations support differences in the distribution and pattern of driver mutations in diseases such as colon cancer that present more in AA patients as compared to CA patients. The etiologic basis for differences between race groups, such as higher rates of KRASmutant tumors in AA colon cancer patients, is not known, and relevance to tumor behavior including effect on chemotherapy responsiveness remains unclear. Importantly, the lack of cell lines, organoids, and/or patient-derived xenograft models representative of disease heterogeneity that reflect differences in disease patterns by race severely limits our understanding of and ability to study the differences in disease behavior between patient populations based on race. This includes assessments of differences in treatment response where much of what is known is based on a few models from CA patients. Overall, the limited availability of racially diverse tissue for research purposes and the lack of therapeutic models hampers the ability to evaluate cancer initiation, progression, and therapy in an inclusive population. This one factor contributes most significantly to gaps in the knowledge of cancer in racially and ethnically distinct populations. This should be seen as a scientific area of high priority needed to reduce the unequal burden of cancer health disparities. Thus, what is extremely important is a more concerted effort to generate diverse in vitro, ex vivo, and in vivomodels. To date, the American Type Culture Collection (ATCC, founded in 1925) has a limited number of cell lines designated as AA or Hispanic American (HA), some organ tumors fair better than other organ tumors. For example, in the ATCC repository, there are no colon cancer cell lines designated as AA. Equally important is the correlation of tumor biology discovery with metadata linking social determinates of health. To meet this need, the establishment of three novel AA CRC cell lines now allows for in vitroand in vivoassessment of compounds and tumor biology which may influence differential chemo-response by race [135]. 3D cell culture in Matrigel©-related protocols may further elucidate these tools for more realistic in vitroexperimentation [135]. Furthermore, our research group and others are developing colon tumor organoids to better recapitulate chemotherapeutic responses representative of the natural tumor environment. Finally, patient-derived xenografts from tumor tissue resected from African American CRC patients are a valuable source of cells for downstream applications like cell culture, primary cell lines, organoids, chemotherapeutic assessment, etc.

Multi-omics assessment combined with accessible tools, the exploration of CRC gene expression, and overall biology will make possible an understanding that can be therapeutically targeted for the maximum benefit of the patient. Discovery and validation methods are provided in Figure 2. Given the multidimensionality of CRC, it is scarcely sustainable to maintain blanket standards of care that have abhorrent side effects with a sizeable chance of failure. Ideally, sampling a patient’s tumor and having a pipeline of treatments optimized for their genetic, epigenetic, and metabolomic profile will be the future of cancer treatment. In a diverse patient population with racial health disparities among other socioeconomic obstacles, research aimed at unpacking these complexities is vital for getting closer to the goal of a personalized approach for healing patients.

Figure 2.

Assessment methods to address differences in response to treatment.