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Treatment Target #1: Forkhead box protein M1 (FOXM1)

FOXM1 has been shown to play a vital role in cell cycle progression, where its
expression peaks at the S and G2/M phases. A recent study has also demonstrated that FOXM1
acts as a regulator for the expression on many G2/M-specific genes, such as:

PLK1 proto-oncogene whose overexpression has been observed in tumor cells; early
trigger molecule for G2/M transition;

Cyclin B2 vital component of cell cycle regulatory machinery; also binds to TGFR2

NEK2 - protein kinase that plays a role in the control of centrosome separation and
formation of bipolar spindles

Centromere protein F (CENPF) encodes a protein that is associated with the

centromere-kinetochore complex; thought to have a role in chromosome segregation
during mitosis

FOXM1 is known to interact with FZR1 (fizzy-related protein homolog 1), which in turn
interacts with CDC27 (cell division cycle protein 27 homolog). These interactions likely lead to
the promotion of cell cycle progression and overcoming cell cycle checkpoints.

FOXM1 has three isoforms, (A, B, and C) the first of which is a gene transcriptional
repressor; the last two are transcriptional activators and are both found to be upregulated in
cancers, just as FOXM1 is shown to be upregulated over tenfold in prostate cancer. The exact
mechanisms by which FOXM1 acts in the formation of cancer are unknown. It is thought that
FOXM1 upregulation promotes oncogenesis from the abnormal impact that is made on its many
roles in progressing the cell cycle and chromosomal/genomic maintenance. Upregulations of
FOXM1 has been shown to induce genomic instabilities such as loss of heterozygosity (LOH)
and copy number aberrations. The downstream targets of FOXM1 are listed above and their
expression results in generally positive cellular proliferation signals, as well as invasion and
metastasis.

FOXM1 seems to present a druggable, rational candidate for therapy because of its
characteristics as a transcription factor. The Fox proteins family all exhibit a special
characteristic, the Forkhead box, a sequence of 80 to 100 amino acids that form a motif and bind
to DNA. This quality seems to suggest that engineering a particle to bind to this specific site and
change the conformation of the protein is possible. However it is also possible that due to
similarity with other Fox proteins, it may be difficult to develop a drug that specifically targets
FOXM1. Possible drug design goals for treating the expression of FOXM1 would be to control
the phosphorylation, acetylation, or ubiquitination of the protein. Inhibition of FOXM1 would
contribute to cancer treatment by inhibiting important components to the cells cycle, and
decreasing the capacity at which a cell can maintain genomic integrity. These effects slow the
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rate of proliferation and induce apoptosis by allowing genome damage to accumulate to such as
extent that the cell cannot continue living. Testing this drug would be straightforward, starting
with in vitro and animal experiments in which tumors are supplanted within mice that both
express FOXM1 and do not express FOXM1, those treated with the inhibiting drug and those
that are not. The testing would then progress to trials that tested for safety in humans, and then
finally to trials that test for efficacy with respect to other, similar drugs.

Treatment Target #2: Aurora kinase A AUKRA,

also known as serine/threonine kinase 15 STK15

STK15 is a serine/threonine-protein kinase that is shown to be overexpressed tenfold in

prostate cancer. It is encoded by the AURKA gene and is implicated with very important
processes during mitosis and meiosis; proper function of this protein is vital to healthy cell
proliferation. STK15 is made active by one or more phosphorylations and has shown peak
activity during G2/M phase transitions in the cell cycle.

STK15 is known to interact with molecule involved with the formation of the mitotic
spindle; STK15 recruits TACC, involved in the stabilization of the central microtubule, Kinesin
5, involved in the formation of a bipolar mitotic spindle, as well as gamma-tubulins, which form
the base structure for centrosomal microtubules to polymerize from. Lack of STK15 results in
the centrosome not accumulating enough gamma-tubulin to healthily enter anaphase. The cell
will still progress through the cycle at this point, but will result in a centrosome that never fully
matures. STK15 is also required for the proper separation of centrosomes after the mitotic
spindle is formed. Without STK15, the mitotic spindle will never separate or attempt separation
and collapse upon itself. Additionally, STK15 also assures the proper alignment of
chromosomes during prometaphase. Finally, STK15 is known to assist in exiting mitosis by
contributing to cytokinesis, the process by which the cytoplasm of the diving cells splits.

Upregulation of this protein, as demonstrated in prostate cancer, should result in the

cancer cell developing a degree of self-sufficiency in orchestrating the cell cycle. Drug design
for this target protein should target the active site at which STK15 is phosphorylated. Though
serine/threonine-protein kinases generally display similarity in terms of shape, the STK15 family
of proteins have a highly conserved C-terminal catalytic domain, suggesting that it could be very
feasible to manufacture a particle that would bind to that domain and inhibit STK15 expression
by competitively inhibiting, or perhaps allosterically inhibiting, the active site at which STK15 is
phosphorylated. It is important to consider that this protein is also normally regulated by p53, so
targeted therapy in conjunction with p53 promotion may prove very effective.

Inhibiting this protein would fight cancer progression by, again, limiting cell
proliferation, and also allowing for more genome damage to occur as a result of the proteins
involvement in chromosome separation. STK15 has also been implicated in the mesenchymal-
epithelial transition, meaning that inhibiting this protein would also slow the rate at which the
cancer cells metastasize.
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Again, this drug could be tested using the model discussed for the first treatment target.
Human tumor xenografts could be performed on mice that are separated into groups that do
express STK15 at normal levels, have inhibited expression of STK15, and mice that overexpress
STK15. This experiment would demonstrate and verify the effect STK15 has on tumor
progression and cancer development. Next, the drug should be tested for safety in humans, and
then finally it should be tested for efficacy with respect to other, similar drugs.

Treatment Target #2: Enhancer of zeste homolog 2 EZH2

EZH2 is a histone-lysine N-methyltransferase enzyme. It functions primarily as a gene

suppressor and is encoded by the EZH2 gene and is also known to participate in DNA
methylation. EZH2 is the enzymatic component of Polycomb Repressive Complex 2 (PCR2),
which regulates differentiation. It methylates PCR2 and also forms a complex with the proteins
EED, SUZ12, JARID2, AEBP2, RbAp46/48, and PCL.

In prostate cancer, EZH2 is overexpressed by more than tenfold. EZH2 overexpression

has been commonly implicated to many forms of cancer, as EZH2 inhibits genes that are
involved in suppressing tumor development. This proteins has been shown to play a vital role in
development, cell differentiation, transcription repression, and transcription activation, though
the exact mechanisms by which it does so for all functions are unknown.

EZH2 is typically unexpressed in healthy adults. It is known only to be found in very

actively dividing cells, such as those present in embryonic and fetal development. Inhibition of
this protein has been reported to cause shrinkage in malignant tumors, due to its action as a gene
suppressor that may silence tumor suppressor genes. EZH2 activity has been shown to be
regulated by phosphorylation of threonine and serine residues. It has also been shown that EZH2
activity is reduced by acetylation and methylation.

The efficacy of a targeted treatment for EZH2 is dubious, however, as there is no

consensus as to whether EZH2 overexpression is the cause or consequence of cancer. It does
however, show promise in druggability, as it is found only in dividing cells and is a member of
the SET domain family. The SET domain is a distinct shape on these molecules that might allow
for a targeted treatment that allows for a particle to bind to this domain and inhibit EZH2
activity, reducing the suppression of tumor suppressor genes. These qualities imply that targeted
treatment for EZH2 would be highly specific to certain groups of cells and more readily affect
cancer cells as opposed to healthy cells.

Testing for a targeted drug therapy for EZH2 would probably first verify that EZH2
expression does in fact have an effect on cancer development. Then it may be tested on nude
mice, using human tumor xenografts to examine whether or not the normal expression,
overexpression, and under expression of EZH2 has any effect on tumor size. The drug trials
would then move on to using EZH2 on humans to test for safety and adverse side-effects.
Finally, EZH2 therapy would be tested for efficacy with respect to other cancer therapies.
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Case Study:
A 71-year old male seeks attention after developing difficulty and pain during urination. In
addition, the patient complains of intermittent pain in his lower back. His physician performs a
digital rectal exam and notes a lump on the prostate. Blood tests for the prostate specific antigen
(PSA) reveal elevated levels (12ng/ml) of this marker. A transrectal ultrasound is performed,
yielding the results seen in the following image. A biopsy and a full body CT scan were
subsequently performed. The pathology of the biopsy confirmed carcinoma of the prostate.
The CT scan indicated metastases
to the bone.

The article Comprehensive Gene Expression Analysis of Prostate Cancer uses gene array
technology to determine the expression levels of genes in prostate cancer. In keeping with the
idea of a more rational form of treatment for cancer, you will propose individualized, targeted
cancer therapies for the treatment of the prostate cancer patient. Assume that the gene array data
detailed in Table 2 of the paper are the data obtained for this patient.

Your task:
Choose 3 targets for therapy considering the gene array data obtained from the patients tumor.
For each target,

1) Explain the role of the target in cancer and its role in normal cells
2) Explain the rationale for choosing the target
3) Propose a means for development of a drug to this target and explain how the drug will work
to affect the target.

Rules of the project:

1. One of your targets must be new (meaning it is not a molecule that we have studied in
class).
2. Choose diverse targets for therapy. Be cognizant of the intricacy of tumor formation: 1)
multi-step tumorigenesis, 2) angiogenesis, 3) metastasis, 4) genome instability, 5)
complexity of tumor structure.
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3. For drug development, you need to be aware that certain features of molecules make
them preferred for targeting. You need to weigh these features and indicate how these
affected your decision in the rationale section.

Grading:
Your project will be graded based on the specifications listed below.
Your project and portfolio are due by the beginning of our final exam period in finals week.
Final Project Specifications:
Choice of targets
One of your targets must be new (meaning it is not a molecule that we have studied in
class).
Three targets were chosen for therapy.
The 3 targets are diverse in nature (target different mechanisms of cancer).
Targets are chosen from article on gene expression in prostate cancer.

Explain role of target #1 in normal cells

The role of the target in normal cells is described.
The cell signaling processes surrounding the target are discussed.
Specific interactions of the target with other molecules are discussed.
The downstream targets are discussed.
The overall outcome of the pathway discussed.

Explain role of target #1 in cancer

The nature of the change (mutation, LOH, promoter methylation, etc.) leading to
cancer is discussed.
The effect of the change regarding the target is described.
The changes are linked to a facet of cancer (tumorigenesis, angiogenesis, etc.).

Rationale for choosing target #1

The target was chosen for ease of drug design (has catalytic cleft or a specific domain
for protein-protein interactions).
A rationale for this particular target is presented.
Features (positive and negative) of target are outlined.
Broader implications of affecting target on cancer discussed.

Proposal for drug design to affect target #1

A proposal for drug design is presented.
A specific component of the target molecule is specified.
A description of how the component would be disrupted/affected is described.
Discussion of testing of drugs included.

Explain role of target #2 in normal cells

The role of the target in normal cells is described.
The cell signaling processes surrounding the target are discussed.
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Specific interactions of the target with other molecules are discussed.
The downstream targets are discussed.
The overall outcome of the pathway discussed.

Explain role of target #2 in cancer

The nature of the change (mutation, LOH, promoter methylation, etc.) leading to
cancer is discussed.
The effect of the change regarding the target is described.
The changes are linked to a facet of cancer (tumorigenesis, angiogenesis, etc.).

Rationale for choosing target #2

The target was chosen for ease of drug design (has catalytic cleft or a specific domain
for protein-protein interactions).
A rationale for this particular target is presented.
Features (positive and negative) of target are outlined.
Broader implications of affecting target on cancer discussed.

Proposal for drug design to affect target #2

A proposal for drug design is presented.
A specific component of the target molecule is specified.
A description of how the component would be disrupted/affected is described.
Discussion of testing of drugs included.

Explain role of target #3 in normal cells

The role of the target in normal cells is described.
The cell signaling processes surrounding the target are discussed.
Specific interactions of the target with other molecules are discussed.
The downstream targets are discussed.
The overall outcome of the pathway discussed.

Explain role of target #3 in cancer

The nature of the change (mutation, LOH, promoter methylation, etc.) leading to
cancer is discussed.
The effect of the change regarding the target is described.
The changes are linked to a facet of cancer (tumorigenesis, angiogenesis, etc.).

Rationale for choosing target #3

The target was chosen for ease of drug design (has catalytic cleft or a specific domain
for protein-protein interactions).
A rationale for this particular target is presented.
Features (positive and negative) of target are outlined.
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Broader implications of affecting target on cancer discussed.

Proposal for drug design to affect target #3

A proposal for drug design is presented.
A specific component of the target molecule is specified.
A description of how the component would be disrupted/affected is described.
Discussion of testing of drugs included.

General project requirements

Appropriate biological terminology is used.
Writing is clear and concise.
Proposal is written in an organized and rationale format.