Colon Cancer: Genetics, Cell Lines (HCT116, HT-29) & Therapeutic Use

This case study examines colon cancer, focusing on the genetic and molecular mechanisms that differentiate normal cells from cancerous ones. It discusses the roles of proto-oncogenes, oncogenes, and tumor suppressor genes in cancer development, highlighting the importance of DNA repair genes. The study also explores colon cancer cell lines, specifically HCT116 and HT-29, detailing their characteristics, mutations, and applications in drug screening and therapeutic research. HCT116, with its KRAS proto-oncogene mutation, is used to study tumorigenicity and gene therapy, while HT-29, derived from a Caucasian female, serves as a model for intestinal cell differentiation. The analysis includes the diagnosis of colon cancer through colonoscopy samples and the significance of understanding cell line phenotypes for biomedical research. This document, contributed by a student, is available on Desklib, a platform offering AI-powered study tools and a wealth of academic resources.

Running head: CANCER
Cancer
Name of the Student
Name of the University
Author note

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Cancer
Cancer results from a cascade of molecular pathways that alter the characteristics of
normal cells. While understanding the biology of cancer, it is caused when cells inside the body
accumulate due to genetic mutations that interfere with normal cell properties. Cancer cells
prevent the invasion and cell overgrowth where mutated cells divide and grow without any
specific signals (De Martel et al. 2012). As these cells divide, they gain new characteristics
including structural changes, new enzymes production and decrease in cell adhesion. This
heritable change makes the cells and its progenies to grow and divide inhibiting the growth of the
surrounding cells. As a result, they spread and start invading cells in proximity. Cancer cells are
malignant in nature that spread or invade to other parts of the body through blood or lymph
forming new tumours distant from the original site. Unlike these tumours, benign tumours do not
invade or spread to other nearby tissues and large (Meacham and Morrison 2013).
Difference between normal cells and cancer cells
Cancer cells differ from normal cells as they have uncontrollable growth rate and
invasive in nature. Normal cells are distinct and specialized to perform specific functions where
cancers cells are not specialized making them divide unstoppably. Cancer cells grow
continuously forming a cluster called tumour having growth factor proteins that make them grow
continuously. Another difference between cancer and normal cells is that the latter does not
responds to signals to top growing. Moreover, they do not repair or undergo apoptosis or die
when they get old or damaged. Cancer cells lack adhesion molecules that make them float and
travel to distant body arts via bloodstream or lymph gaining the ability to metastasize
(Vogelstein et al. 2013). When they reach new regions like lungs, lymph nodes, bones or liver,

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they start growing and form tumours at the secondary locations. In contrast to normal cells, there
is variability in cancer cell size as they are larger with abnormal shape, nucleus or cell
organelles. The nucleus appears darker and larger containing excess DNA having abnormal
chromosomes arranged in disorganized manner.
Genetics of cancer
Only small proportion of 35,000 human genes is associated with cancer and alterations in
these genes are associated with various cancer forms. This malfunctioning is broadly divided
into three groups: proto-oncogenes, oncogenes and tumour suppressor genes. Proto-oncogenes
in normal cells code for proteins that send signals for cell division to the nucleus initiating signal
transduction pathway. This cascade comprises of membrane receptors for signalling and
intermediary proteins that carry signal to cytoplasm and transcription factors activate cell
division genes in the nucleus. However, altered versions of proto-oncogenes give rise to
oncogenes activating signals stimulating uncontrolled growth. Proto-oncogenes like RAS, MYC,
WNT, TRK and ERK acquire activating mutation that form tumour-inducing molecule called
oncogene through three activation methods involving gain-of-function mutation (Hnisz et al.
2016). Point mutations occur in proto-oncogenes result in constitutively protein product. Gene
amplification or localized reduplication of DNA segment in proto-oncogene also leads to
overexpression of protein. Chromosomal translocation results in growth-regulatory gene that is
controlled by a different promoter resulting in inappropriate gene expression.
Tumour suppressor genes also play an important role in cell growth and division. They
act to inhibit cell proliferation and development of tumour. However, these genes lose control or
gets inactivated that remove negative regulators in cell proliferation and contribute to abnormal

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tumour cell proliferation (Yates and Campbell 2012). Mutations occur in DNA repair genes like
mismatch repair (MMR) genes, nucleotide excision repair (NER) group, DNA crosslink repair
that results in inherited cancer syndromes. DNA repair mechanism is the backbone of survival;
however, decreased efficiency or inactivation occurs by epigenetic gene activation mechanism
that affects DNA repair activity of the genes (Jeggo, Pearl and Carr 2016).
Types of cancer
Carcinoma cancers begin in tissue or skin that covers the surface of glands or internal
organs forming solid tumours. Examples comprises of breast cancer, prostate cancer, colorectal
and lung cancer. Sarcoma begins in the connective tissue that connects and supports the body
developing in nerves, muscles, fat, joints, tendons, cartilage, blood vessels or bone. Leukaemias
are blood cancers that begin when there is change in structure and uncontrollable growth occurs
in blood cells. The four main types of leukaemia are acute and chronic lymphocytic leukaemia,
acute and chronic myeloid leukaemia. Lymphomas are cancer that begins in lymphatic system
causing Hodgkin and non-Hodgkin’s lymphoma. Melanomas are cancers that begin in cells that
form melanocytes that are specialized cells synthesizing melanin, pigment that impart colour to
the skin causing skin cancer (Sandoval and Esteller 2012). Colon cancer is also a type of
adenocarcinoma that is discussed in the subsequent section.
Colon cancer
Colon cancer is the large intestine cancer that makes up the final part of the digestive
tract. Mostly, colon cancer begins as a benign or non-cancerous clump of cells known as
adenomatous polyps. These polyps are small and show few symptoms. They comprise of excess
abnormal and normal appearing cells in the glands that cover colon’s inner wall. These abnormal

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growths later enlarge and degenerate to become adenocarcinomas. This cancer generally occurs
before the age of 40 years and due to lifestyle factors. More than 75-95% colon cancers occurs in
individuals with no or little genetic risk and out of which 10% is linked to sedentary lifestyle and
addictive behaviour (Kandoth et al. 2013). It originates from epithelial cells that lines colon of
the gastrointestinal tract due to mutations in Wnt signalling that enhance signalling activity.
Colon cancer are acquired or inherited that occur in crypt stem cell of the intestine.
Adenomatous polyposis coli (APC) gene is also polyposis 2.5 deleyed protein that is encoded in
humans by APC gene. This protein is a negative regulator of beta-catenin concentrations and E-
cadherin that play an important role in cell adhesion. This protein prevents the beta-catenin
protein accumulation and as a result, it is accumulated to high levels that translocated to nucleus,
DNA binding and activates proto-oncogenes transcription (Burrell et al. 2013). Mostly, APC
gene is important for cell differentiation and renewal, but at inappropriately high levels, it causes
cancer. Apart from Wnt signalling, other mutations also occur that make cells cancerous like p53
gene that monitors cell division. Inherited gene mutations increase the risk of colon cancer that is
present in two most common forms. Hereditary nonpolyposis colorectal cancer (HNPCC) or
Lynch syndrome develop in individuals before the age of 50 years. Familial adenomatous
polyposis (FAP) is a disorder that greatly increases the risk of colon cancer and develops polyps
in the colon and rectum lining (Isik et al. 2014).
Diagnosis of colon cancer is performed through sampling of colon obtained during
colonoscopy that depends on lesion location. The cell line acquires mutation where it transforms
from benign to invasive epithelial cancer cells. To study the phenotype of colon cancer, it is
important to study cell lines that are considered efficient biomedical research tools that
emphasize on genotype characterization and authentication (Kreso et al. 2014). There are 24-

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colon cancer cell-lines that vary in growth characteristics and appearance. They are used to study
the biology of colon cancer and test treatments. The established cell lines maintain and represent
the genetic diversity of the primary colon cancers. DNA copy number and exome mutation
spectra in colon cancer cells closely resemble to hypermutation phenotypes defined by defective
DNA polymerase and DNA mismatch repair, proofreading deficiency and concordant mutations
altered in Wnt, p53 pathways (Cayrefourcq et al. 2015).
Colon cancer cell lines
Among all the cell lines in colon cancer, HCT116 and HT-29 is human colon cancer
cells that are used in drug screening and therapeutic research. HCT-116 has mutation in KRAS
proto-oncogene in codon 3. They are considered suitable transfecting agents or the gene therapy
and research. These cell lines possess epithelial morphology that can be used to study the
tumorigenicity. They have adherent properties having epithelial morphology having posttive
control for PCR mutation assays present in codon 13 (Wang et al. 2013). When these cell lines
are transducted with viral vectors that carry p53 gene, there is arresting of HCT116 in the G1
phase. Colony proliferation of HCT116 was inhibited by P85/5-Fu to study the proliferation of
colon cancer and corresponding inhibitors. This cell line has two important variations: Insp8
gene having large expression and one without it. This gene plays a role in the metabolism of
cell’s energy processing that can in turn also affects the cell phenotype (Sun et al. 2012).
The doubling time of HCT-116 is ~18 hours and is suitable for in vivo and in vitro
experimentation in life studies where it forms tumours and metastasize followed by implantation
of cells (Kim, Lee and Kim 2013). These cell lines are also beneficial in evaluating the effects of
therapies and drugs available with different reporters and facilitate multi-modality imaging. For

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high and constitutive expression of HCT-116 reporter proteins, generation of cell lines are
carried out by lentiviral vector transduction. These vectors are used for carrying out
transductions that are self-inactivating (SIN) vectors where there is deletion of viral promoter
and enhancer. This results in increase in the biosafety of lentiviral vectors through prevention of
mobilization of competent viruses’ replication. These cancer cell lines are genotypic in nature
that results in reduced responsiveness over time that ensures performance and stability of the
cancer cell lines (Sahlberg et al. 2014). This cell line does not express or differentiates CDX1 or
sub-populations of cells having great tumour-forming capacity. This suggests that HCT-116
contains cancer stem cells (CSCs) subpopulations that are characterized by cell surface markers
and morphology of colony having self-renewal ability and differentiation into multiple lineages.
HT-29 was initially derived from Jorden Fogh, 44-year-old Caucasian female in 1964 is
also epithelia in nature having adherent properties. They have a unique model that can be used to
study molecular mechanisms of differentiation of intestinal cells. Under appropriate culture
media conditions, these cell lines can be manipulated to express enterocyte differentiation. This
is the reason HT-29 is considered pluripotent cells that can be used to study molecular and
structural events that are involved in colon cancer cell differentiation. This cell line forms tight
monolayer that exhibit similarity to the enterocytes in the small intestine. These cell lines
increase the production of p53 tumour antigen having a mutation at the 273 position that results
in replacement of arginine to histidine (Bogaert and Prenen 2014).
HT29 cell line is used in preclinical research for the differentiation ability and thus
mimics real colon cells in vitro successful for research of epithelial cells. HT-29 terminates
differentiation into enterocytes replacing glucose by galactose in the cell culture medium.
Moreover, these cells have induced differentiation property that is galactose-mediated that causes

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adherens junction strengthening. These cell lines also proliferate in cell culture media that lack
growth factors with 4 days doubling time, although it can reduced by using fetal bovine serum
(FBS). They have high glucose consumption and remain undifferentiated in standard medium
(Mouradov et al. 2014). They grow in multi-layer comprising of unpolarised differentiated cells
that does not possess characteristics of normal intestinal epithelia cells and low amount of
hydrolases. Biochemical markers physiologically and morphologically characterize the polarised
phenotype of HT-29 colon cancer cells (Martínez-Maqueda, Miralles and Recio 2015). The
differentiation is influenced by growth related factors that starts after confluence forming a
monolayer and tight junctions. The brush border of HT-29 contains proteins that are present in
normal intestinal microvilli like villin. Moreover, these cells also express hydrolases that are as
small intestine; however, enzymatic activity is much lower than normal intestine with no lactase
expression.
HT-29 show relevance to in vivo as there is similar protein expression as human
intestinal cells. The significant differences in metabolic and transporters gene expression from
the human intestinal cells also affect suitability of HT-29 in showing in vivo permeability.
According to Cancer Genome Atlas Network (2012), the expression of 377 genes in this cell line
used as in vitro models in epithelium showed that HT-29 differentiation has similarity with
human colonic tissues and are not different. However, there are significant advantages and
disadvantages of using HT-29 cell line in vitro as summarised by (Wilding and Bodmer 2014).
The differentiated phenotype of this cell line is similar to enterocytes of small intestine in regards
to structure, cell differentiation process and brush-border hydrolases. The amount of villin
expressed in HT-29 differentiated cells shows similarity with freshly prepared colonocytes. On a
contrary, they are malignant cells that have high glucose consumption and glucose metabolism

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impairment. Due to brush-border hydorlases, they do not show any similarity with normal
enterocytes. Altogether, HT-29 colon cancer cell lines are considered valuable model for
studying the biology of colon cancer cells (Calon et al. 2012).
Multi-drug resistance in cancer
Cancers generally develop resistance to the chemotherapies and there is an increased
prevalence of drug resistant cancers. The ability of cancer cells to become resistant is
functionally and structurally unrelated to anti-cancer drugs called multi-drug resistance (MDR).
Chemotherapy is the treatment given to the patients diagnosed with locally, advanced and
metastasized cancer. This poses challenge in administering drug dosage that minimizes treatment
toxicity and maximizes efficacy. MDR is multifactorial and follow cellular pathways that are
involved in drug resistance in cancer. MDR in cancer is a mechanism where they develop
resistance to chemotherapy that results in minimization of cell death and drug-resistant tumours
expansion (Kathawala et al. 2015). This problem affects the treatment of cancers posing major
challenge to the researchers. This resistance occurs against anticancer drugs and as a result, there
is decreased drug uptake, activation of detoxification system, increase in drug efflux, and evasion
of apoptosis (drug-induced) and DNA repair mechanism activation.
MDR either acquired or inherent exist against anticancer drugs developed through
multiple mechanisms. Drug inactivations that occur in vivo undergo complex mechanisms
where drugs interact with different proteins that modify or partially degrade the other molecules
leading to activation. In such cases, anticancer drugs undergo metabolic activation that acquire
efficacy. Due to decrease in drug activation, cancer cells may develop resistance to
chemotherapies. Many anticancer treatments demand metabolic activation and therefore cancer

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cells may develop resistance through change in proteins that are related to apoptosis. For
example, p53 gene is mutated in half of cancers where deletion or mutation of this gene results in
non-functional form that results in MDR. On a contrary, p53 regulators inactivation like
apoptotic protease activating factor 1 (Apaf-1) and caspase-9 and cofactors can result in drug
resistance (Kibria, Hatakeyama and Harashima 2014).
Another mechanism is alteration of drug targets takes place due to modifications in
expression levels or mutations. This can be explained in a manner where efficacy of a drug is
influenced by molecular target and its alterations. One form of such mechanism is that in certain
cases, topoisomerase II is targeted by anticancer drugs that prevents under or super-coiling of
DNA. The complex that is formed between topoisomerase II and DNA is transient, however,
these drugs leads to DNA damage, DNA synthesis inhibition and halting of mitosis. Moreover,
cancer cell lines can also develop resistance to this enzyme through mutations that takes place in
topoisomerase II gene. Another mechanism that can cause MDR in cancer is through signalling
kinases like epidermal growth factor receptor (EGFR) family and downstreaming of signalling
proteins like Raf, Ras, Src and MEK (Wu et al. 2014). These kinases are active in some type of
cancers promoting uncontrolled cell growth. In such circumstances, over-activation of these
kinases is caused by mutations that may also occur due to over-expression of genes. Apart from
alterations that occur in drug targets, the resistance occur through alteration in process of signal
transduction that mediates activation of drugs.
The mechanism that is most commonly studied in MDR in cancer is drug efflux that
involves reduction in drug accumulation through efflux enhancement. ATP-binding cassette
(ABC) transporter family proteins are transmembrane proteins that are classified into two
domains having highly conserved binding domain for nucleotides and variable transmembrane

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domain. When a substrate binds to the transmembrane domain, there is hydrolysis of ATP
occurring at nucleotide binding site that acts as a driving factor for conformation change pushing
the substrate out of the cell. ABC transporters induce efflux considered as normal process;
however, it is also a mechanism for MDR in cancer cells. There are three transporters called
multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1) and
breast cancer resistance protein (BCRP) that are responsible for MDR in cancer (Zahreddine and
Borden 2013). These transporters have broad specificity for substrates and as a result, efflux the
xenobiotics from the cells. Therefore, the cancer cells are protected from first line
chemotherapies inducing MDR.
DNA damage repair also performs a role in developing MDR. Chemotherapies indirectly
or directly damage DNA and its response mechanisms that can reverse the damage that is
induced by cancer. For example, few anti-cancer drugs cause DNA crosslink that result in
apoptosis. On a contrary, resistance to these drugs arises due to homologous recombination and
nucleotide excision repair that reverses the effect of this anticancer drug property. Moreover,
impairment or dysregulation of DNA damage response (DDR) due to epigenetic silencing or
mutations can result in increased resistance and failure to chemotherapy (Gillet and Gottesman
2010).
Cell death inhibition is another mechanism where recombinant forms of TNFs related
apoptosis-inducing ligand (TRAIL) induce apoptosis through caspase-8 activation. However,
results showed that prolonged use of these drugs causes resistance and they need to be used in
combination with other cytotoxic drug so that it kills the cancer cells in vulnerable states
(Zahreddine and Borden 2013).

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Epithelial-Mesenchymal Transition and Metastasis (EMT) is a process where solid
tumors metastatic causing angiogenesis. EMT plays an important role in drug resistance
development depending on the metastatic tumor grade defined by EMT degree and level of
differentiation. MDR that occur in cancer cells during differentiation and its signaling process
are essential for the mechanism of EMT. For example, in colon cancer, there is increased integrin
αvβ1 expression that regulates positive transforming growth factor β (TGFβ) expression, EMT
expression and acts as survival signal for the cancer cells against anticancer drugs (Housman et
al. 2014).
Cancer cell heterogeneity is a mechanism involved in MDR where fraction of cells
presents in heterogenous populations possess stem cell properties and drug resistant. Moreover,
small fraction of adult stem cells (ASC) also has MDR capabilities. Cancer treatment kills drug
sensitive cells and result in drug resistance where cancer cells survive, grow, expand and
metastasize. Drugs that may go into circulation and form secondary tumours in the distant organs
in the body kill some of these resistant cells. As a result, heterogeneity is witnessed in cancer in
both solid tumours and circulation (Zahreddine and Borden 2013).
Drug repurposing
Drug repurposing is commonly referred to as therapeutic switching or re-tasking
encompasses the application of drugs and compounds that are known, with the aim of treating
particular diseases. It has been identified as a promising pharmaceutical strategy that effectively
reduces the resources required for development of therapies for certain diseases. The process also
amplified the probability of the drug entering the market from phase I, for new indications
(Strittmatter 2014). This approach of drug repositioning is found to primarily capitalize on and