Focal adhesion kinase signaling in metastasis and breast cancer treatment Nimwegen, M.J. van

Focal adhesion kinase signaling in metastasis and breast cancer treatment

Nimwegen, M.J. van

Citation

Nimwegen, M. J. van. (2007, March 21). Focal adhesion kinase signaling in metastasis and breast cancer treatment. Division of Toxicology,

Leiden/Amsterdam Centre for Drug Research, Faculty of Mathematics and Natural Sciences, Leiden University. Retrieved from

https://hdl.handle.net/1887/11415

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1

General Introduction:

Breast Cancer Development,

Metastasis and Treatment

1 I

.

This thesis focuses on the role of focal adhesion kinase (FAK) in the processes of tumor formation, metastasis and anti-cancer drug sensitivity. In this first chapter the general aspects of breast cancer, its etiology and current treatment regimens are described, without the intention to critically discuss these various aspects. After this brief introduction, this chapter concludes with the outline of this thesis. The involvement of FAK in the development and progression of cancer is critically discussed in Chapter 2.

1.1 I

.

Breast cancer is the second most common cancer among women. Despite earlier detection of the disease and improved anti-cancer therapies, it is still the second leading cause of cancer related deaths among women. According to the World Health Organization, world-wide, more than 1.2 million people, 1% male, will be diagnosed with breast cancer this year. One out of every seven women will develop an invasive form of breast cancer during her life.

Like many diseases, there are multiple risk factors for the development of breast cancer (reviewed in [1]). Among lifestyle related factors are alcohol, obesity and oral contraceptives. In contrast to other cancers, such as lung cancer, in which there is a very strong correlation between the occurrence of the disease and smoking, with breast cancer the non-lifestyle risk factors are far more important.

These include aging and genetic predisposition, but the most important risk factor, increasing the chance 100 times, is being a woman. This is not because women have more breast tissue, but because female breast cells are continuously exposed to high concentrations of the female hormones estrogen and progesterone, providing growth-promoting effects.

1.2 D

(

)

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Hallmark of cancer is the uncontrolled proliferation of cells. Cancer cells, unlike

normal cells, ignore signals to specialize, stop dividing and die. The disturbed life cycle

of cancer cells is caused by one or multiple mutations, either somatic or germline

(inherited), in the DNA of the cells. Somatic mutations, occur during the life-span of

a cell and can be caused by exogenous stimuli e.g. chemicals and UV radiation. These

mutations may be rapidly repaired by the cell its gene-repair mechanisms, or may be

reason for the cell to commit suicide (undergo apoptosis) or to be killed by cells of the

immune system. Thereby, growth of cells with mutated DNA is prevented. However,

when mutated cells escape these repair/killing routes, uncontrolled division can take

place and a tumor is formed. About 5 to 10% of all breast tumors are hereditary as

a result of mutations in the DNA. Inherited mutations of genes that increase the risk

of breast cancer include BRCA1, BRCA2, p53, ATM and PTEN (reviewed in [2]). Only cells that obtain multiple mutations (somatic, germline or a combination of both) are thought to be able to eventually form tumors.

2 T

. 2.1 S

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The most effective way to treat breast cancer is surgical removal of the tumor. The stage of the disease (see paragraph 2.4) determines whether the surrounding tissue, the whole breast or the lymph nodes should be removed in addition to the primary tumor. Importantly, all individual tumor cells have to be removed to prevent outgrowth of residual cells into new tumors. Therefore, surgery is almost never the sole therapy in (breast) cancer.

2.2 H

.

As mentioned previously, the continuous exposure of the female breast cells to the hormone estrogen is a high risk factor for the development of breast cancer. Almost 75% of all breast cancers are positive for the estrogen receptor [3]. In these cells, binding of estrogen to the estrogen receptor provides proliferation signals and therefore the uncontrolled division of these cells is (at least partly) dependent on estrogen. Hormonal therapy of estrogen-positive breast cancer consists of blocking these receptors, without activating them, thereby inhibiting the division of the cancer cells [4]. The most well known anti-estrogen drug used in breast cancer therapy is tamoxifen.

2.3 I

:

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A third method that is often used to treat breast cancer is the induction of cell cycle arrest and/or apoptosis in cancer cells by either irradiation or chemotherapy.

Apoptosis is a tightly regulated and complex process of cell death. Upon (sub)cellular injury, a cell decides whether the damage can be repaired or whether it is better to undergo apoptosis. The latter involves activation of the caspases: executioners of the apoptotic process (Fig. 1). Activated caspases cleave a large number of structural and signalling proteins, eventually resulting in fragmentation of the nucleus and the formation of apoptotic bodies. These bodies can be taken up by neighbouring cells or by cells of the immune system. In addition to induction by exogenous stimuli like irradiation and chemotherapy, apoptosis is also involved in various physiological processes, including embryogenesis, tissue homeostasis and maturation of the immune system. During radiation therapy, ionizing radiation is applied locally at the site of the tumor [5], while chemotherapy is administered systemically.

Chemotherapeutic drugs (or cytostatics) act primarily on dividing cells and, therefore,

in addition to the dividing cancer cells, cells of the digestive tracts, hair follicles and

cells of the immune system are also affected. This results in classic side-effects like nausea, hair loss and increased vulnerability towards pathogens.

Chemotherapeutic drugs can be divided into several groups on how they act on the cancer cells [6]. Alkylating agents, anthracyclines, topoisomerase inhibitors and mitotic inhibitors will be briefly discussed. Alkylating agents bind directly to DNA and thereby prevent the cancer cell from reproducing. These agents are not cell phase-specific and include cisplatin, carboplatin and melphalan. Anthracyclines interfere with enzymes involved in DNA replication and also work in all phases of the cell cycle. A major concern is the effect they can have on heart muscle cells. Examples include daunorubicin and doxorubicin (Adriamycin). Topoisomerase inhibitors interfere with enzymes called topoisomerases, which are important in DNA replication.

Figure 1: Intracellular apoptotic signaling. Exposure of cells to chemotherapeutic agents or radiation results in DNA and/or mitochondrial damage. Cells can either repair the damage, undergo cell cycle arrest or activate its apoptotic machinery via the release of cytochrome C from the mitochondria, followed by the activation of the executioner caspases. Bcl-2 (partly regulated by the EGFR-PKB pathway) can prevent cytochrome C realease and the pan-caspase inhibitor zVAD-fmk can prevent caspase activity.

Examples of topoisomerase II inhibitors include etoposide (VP-16) and teniposide.

Finally, mitotic inhibitors are plant alkaloids and other compounds derived from natural products. They can stop mitosis or inhibit enzymes from making proteins needed for reproduction of the cell. These drugs act during the M phase of the cell cycle and examples include the taxanes (paclitaxel, docetaxel) and the vinca alkaloids (vinblastine, vincristine, and vinorelbine). Due to the upregulation or constitutive activation of certain proteins, pathways that are protective against apoptosis are activated. This can result in the reduced the efficacy of chemotherapeutic agents.

Several protein tyrosine kinases (PTK, see also below) are implicated in this reduced sensitivity of cancer cells towards cytostatics. We studied the role of PTK focal adhesion kinase (FAK) in the response of cancer cells to chemotherapy.

2.4 (N

)

.

As a result of extensive research on the development of (breast) cancer, new

molecular targets have been identified as potential drug targets. One interesting

group of proteins that seems to be highly involved in tumorigenesis as well as

metastasis are the so-called tyrosine kinases. These proteins, which can be either

localized as receptors at the cell membrane or in the cytosol, are proteins capable of

phosphorylating downstream effector proteins on tyrosine residues. Phosphorylation

can lead to (in)activation but can also result in targeting of these proteins to specific

places in the cell. Several tyrosine kinases are either upregulated or mutated in

cancer and multiple inhibitors of tyrosine kinases are currently under (pre)clinical

investigation (reviewed in [7]). In about 30% of the patients with invasive breast

cancer amplification of the gene that encodes the HER2/neu receptor tyrosine kinase

is seen. This kinase is involved in signal transduction pathways that lead to cell

proliferation and differentiation. Clinical studies show a significant correlation between

HER2/neu gene amplification and reduced survival relative to patients with normal

receptor levels. This led to the development of Herceptin: an anti-cancer drug based

on the targeting of a kinase. Herceptin is effective in the treatment of metastatic

breast cancer when used alone, and in combination with standard chemotherapy. In

addition to Herceptin, inhibitors of the EGFR (Iressa) and c-KIT (Imatinib) are already

used as anti-cancer drugs, whereas several other tyrosine kinase inhibitors are

currently under investigation for their use as anti-cancer drug. These new drugs have

been (or are) developed because of the improved insights into the development and

progression of breast cancer. Unravelling the mechanisms of metastasis will provide

additional useful targets to combat the lethal metastases and will lead to the

development of new, or improvement of the current breast cancer treatment. The

non-receptor tyrosine kinase focal adhesion kinase might be such a potential new

target (see Chapter 2 for details).

3 M

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3.1 P

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Several mutations are required for a cell to acquire the ability to grow in the absence of the proper survival and proliferation signals to form a primary tumor. When the tumor is restricted to its primary site, the disease can be relatively easily treated by surgical removal of the tumor. Consequently, most cancer deaths are not caused by the primary tumor, but by the occurrence of distant tumors derived from this primary tumor. The process of the spreading of the tumor cells to distant organs is called metastasis. The metastatic process consists of a large number of steps (Fig. 2) [8]. First of all, cancer cells have to undergo epithelial to mesenchymal transition to be able to escape the primary tumor. This means that the tumor cells loose contact with their neighbouring cells and are then able to migrate. Migrating cells can either transfer to the border of the tumor to invade neighbouring tissue, or they can invade a lymphatic or blood vessel and travel to a different site of the body. During the migration/invasion process the cell excretes enzymes, including so-called matrix metalloproteinases's (MMPs), that degrade the extracellular matrix that is present in, and surrounds the tumor.

Figure 2: Metastasis. The metastatic process starts with the uncontrolled growth of cells (primary neoplasm), followed by epithelial to mesynchymal transition (EMT) to migrate through the extracellular matrix (ECM). To provide the primary tumor with growth factors, angiogenesis is stimulated. Next, metastatic cells invade the newly formed bloodvessels and travel to a distant organ to extravasate and finally proliferate to form metastases.

To detain cells at their right anatomical site, for example kidney cells in the kidney, loss of contact with either neighbouring cells or with extracellular matrix normally induces a specific form of controlled cell death, known as anoikis [9].

Metastasizing cancer cells survive travelling in the lymphatic and blood vessels, and therefore are (at least partly) insensitive towards anoikis. The next step in metastasis is to adhere to and subsequently extravasate the lymphatic or blood vessel at the site of the target organ. There, the cells can immediately start to proliferate and form metastases, or they can go into a state of dormancy, in which the cells are neither dying nor proliferating. Only when these dormant cells are exposed to the right molecular cues, proliferation will occur. Not all primary tumors are able to metastasize and, therefore, metastasis is a very ineffective process. Only a small part of the cells in the primary tumor are able to undergo metastasis and only one percent of the metastasized cells grow out into metastases.

3.2 M

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Metastasis is a very complicated, not yet totally elucidated, process. It requires both the appropriate cues from the environment as well as the right expression and activity of distinct proteins in the cancer cells. In order to progress through the different steps of metastasis, several classes of proteins are overexpressed in cancer cells (reviewed in [8]). Some of the important proteins involved in the separate steps are summarized in table 1. Metalloproteinases (MMPs) are enzymes that degrade the extracellular matrix and are implicated in one of the first steps of metastasis: the invasion through the stroma [10]. Another class of proteins involved in metastasis consists of the chemokine receptors. Expression of the chemokine receptors CXCR4 and CCR7 on breast cancer cells are responsible for the selective target organs (lymph nodes, lung, liver and bone marrow) to which breast cancer often metastasizes [11]. Protein tyrosine kinases are involved in several steps

Metastasis process Molecules involved

EMT Src kinase and other PTK’s Proliferation Growth factor receptors, PTK’s

Angiogenesis VEGF, uPA

Migration MMPs, PTK’s

Survival p53, tyrosine kinases Invasion MMPs, chemokine receptors

Table 1: Some of the important molecules involved in distinct steps of metastasis.

of the metastasis process. These proteins become activated in response to different stimili, i.e. attachment of the cells, growth factors, and subsequently phosphorylate and thereby activate a number of downstream targets. Focal adhesion kinase is one of these PTK's and its role in metastasis is studied in this thesis

3.3 G

.

As previously mentioned, in order to undergo all different steps involved in metastasis a cancer cell needs to have obtained multiple mutations. In the classic theory, these mutations have been obtained during the life-cycle of the cancer cell:

first, genetic changes involved in the disturbed life-cycle occur, followed by additional mutations in genes that are involved in migration and survival. In addition to this multi-step model a quite different mechanistic model is described [12]. In this model, it is suggested that the ability of a cancer cell to metastasize is already determined by the spectrum of mutations that progenitor cells have acquired early in tumorigenesis. Consequence of this latter model is that already at a very early stage in tumor formation, the course of the disease can be predicted. Several studies using patient material have been and are being performed to verify this.

4.1 A

.

One way to provide insights into the molecular pathways involved in tumorigenesis and metastasis is the analysis of patient material. By comparing gene expression profiles of cancerous with normal tissue, molecular targets involved in tumorigenesis can be assigned. Likewise, comparing metastatic tumor tissue with primary tumor tissue might reveal molecules crucial in metastasis. This method provides potential targets that then need to be studied in in vitro or/and in vivo models to confirm their involvement in tumorigenesis / metastasis. This method is not only useful in defining new drugable targets; it can also be very helpful in deciding what kind of therapy would be most effective for the patient. These so-called patient- tailored therapy strategies rely on the comparison of the patient's gene profile with a large set of gene-profiles of patients with known responses to used therapy.

Van 't Veer et al. applied DNA microarray analysis on primary breast tumors of 117

young patients and were able to identify a gene expression signature that was

strongly predictive for the occurrence of metastases early in the disease [13]. These

results are in favour of the theory that the metastatic potential of cancer cells is

already present at early time-points in cancer progression. This strategy can be used

to select patients who would benefit from chemotherapy in addition to surgery. In

addition, genes that are overexpressed in patients with a poor prognosis are

potential targets for the development of new drugs. To study the role of these genes,

in vivo models systems are required. Analysis of patient material revealed increased

levels of FAK in primary tumors and metastases [14, 15, 16]. In this thesis, we used

in vitro and in vivo studies to try to unravel the role of FAK in the different steps of metastasis formation. Furthermore, we used these models to study the role of FAK in the sensitivity of breast cancer cells to chemotherapy.

4.2 M

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Due to the complex nature of metastasis formation, in vivo experiments are indispensable in unravelling this process. Transgenic mice are often used in the study of cancer. In these, putative oncogenes or tumorsuppressors can be overexpressed (or downregulated), either constitutively or inducibly, and spontaneous or chemically induced tumor / metastasis formation can be studied. Although studies involving transgenic mice provided information on tumorigenesis, they have been less successful in replicating advanced cancer. Since metastasis occurs as a result of multiple gene mutations, a particular genetic alteration frequently leads to lower metastastatic rates in transgenic mice compared to metastasis in humans. On the other hand, orthotopic xenografts of human tumors, or tumor cell lines, in nude mice reproduce the histology and metastatic pattern of most human tumors at an advanced stage. In vitro, these cell lines can be genetically manipulated and subsequently xenograft models can be used to molecularly dissect the metastatic process.

Nevertheless, this approach is not useful in the study of the initial stages of tumorigenesis or the contribution of the immune system in this process. A third model is the syngeneic model. In this model a tumor cell line is isolated from a spontaneous or chemically induced tumor, studied or manipulated in vitro and subsequently transferred to animals with the same genetic background. Characteristics of these obtained cell lines, like migration, invasion, proliferation, i.e. processes important in tumorigenesis and metastasis, can be studied in vitro. By combining the in vitro and in vivo data, this model can be used to molecularly dissect the metastatic process, including the contribution of the immune system.

5.1 C

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In this thesis, the mammary adenocarcinoma cell line MTLn3 was used to study processes involved in tumor growth and metastasis and the sensitivity towards anti-cancer drugs. This cell line originates from the MTC cell line, which is derived from a primary breast tumor (13762) of a Fischer 344 rat [17]. After several passages of in vitro culturing, MTC cells were injected into the fat pad of a female Fischer 344 rat.

The occurring lung metastases were dissected, cloned and maintained in culture,

resulting in the metastatic MTLn3 cell line. This cell line has been used, by a large

number of groups, including ourselves, both in vitro and in vivo to study responses to

drug treatment as well as the molecular mechanisms of tumorigenesis and

metastasis. In this thesis, the role of the tyrosine kinase focal adhesion kinase (FAK)

is studied in several processes involved in tumorigenesis and metastasis of the MTLn3

cells in vitro as well as in vivo. In addition, both in vitro and in vivo, the sensitivity towards the anti-cancer drug doxorubicin is studied in relation to the activity of FAK in the MTLn3 cells. In vivo, the syngeneic Fischer 344 rat model was applied.

5.2 D

:

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MTLn3 cells are responsive to anti-cancer drugs [18]. This allows us to study the interaction between FAK activity and the sensitivity towards chemotherapy.

Doxorubicin is commonly used in the treatment of metastasized breast cancer. A large number of studies have already unravelled the mechanism by which this anti-cancer agent induces cell damage. Therefore, we have chosen doxorubicin as a model compound for cytostatics. Doxorubicin is an anthracycline antibiotic that induces apoptosis by blocking DNA synthesis in two different ways. Firstly it acts as an intercalating drug by inserting itself between DNA, thereby inhibiting transcription of DNA in the interphase of the cell division process. Secondly doxorubicin inhibits topoisomerase II, which eventually results in DNA strand breaks. Both ways result in cell cycle arrest followed by apoptosis. In addition, doxorubicin can be reduced to a semiquinone free radical intermediate by complex I of the mitochondrial electron transport chain, resulting in the generation of reactive oxygen species (ROS) [19].

One of the problems in the treatment of cancer is the occurrence of resistance towards the used chemotherapy. In this thesis we study the role of FAK in the sensitivity of MTLn3 cells towards doxorubicin.

5.3 A

.

As mentioned before, in order to form a distant metastasis, a cancer cell has to

migrate out of the primary tumor, intravasate into a blood or a lymphatic vessel,

subsequently survive in the absence of cell-cell and cell-matrix interactions,

extravasate the blood or lymphatic vessel, migrate through the target organ and

finally proliferate to grow out into a full metastasis. During all of these processes,

specific kinases are involved in the concerted activation of distinct signalling

pathways. We hypothesised that the protein tyrosine kinase FAK plays a crucial role

in one or multiple of the processes involved in the formation of metastases. Therefore,

the overall aim of the studies described in this thesis was to investigate the role of the

non-receptor protein tyrosine kinase FAK in the distinct processes involved in

tumorigenesis and metastasis and to unravel the involved downstream signalling

pathways. Moreover, the potential of a combined therapy of the inhibition of FAK and

exposure to the cytostatic doxorubicin was tested, as well as dissection of the

intracellular events downstream of FAK.

5.4 O

.

In chapter 2 we summarize the evidence of the importance of FAK in cancer and we

discuss the potential of FAK as an anti-cancer drug. Chapter 3 describes the

cooperation of FAK and the survival mediating protein kinase B (PKB) in the

suppression of doxorubicin-induced apoptosis of MTLn3 cells in vitro. The depletion of

NK cells resulting in the optimization of a syngeneic MTLn3-Fischer 344 experimental

metastasis model is described in chapter 4. In chapter 5 first the creation and

characterization of an inducible HA-FRNK expressing cell line is described. Using this

cell line, the role of FAK in processes involved in tumorigenesis and metastasis is

discussed, providing evidence for a crucial role of FAK in the early phase of

experimental metastasis. The efficacy of the combined therapy of the

chemotherapeutic agent doxorubicin and expression of HA-FRNK in the treatment of

primary tumors and experimental metastases is discussed in chapter 6. Next, we

studied FAK mediated pathways that may be responsible for the observed

FRNK-mediated effects on tumor growth, metastasis and survival. Gene expression

profiling using DNA micro array technology of control and HA-FRNK expressing MTLn3

cells is discussed in chapter 7. Finally, the data is critically discussed in chapter 8 in

view of FAK as a target in the development of new anti-cancer therapies.

R

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