3. Introduction
3.1 Breast Cancer and Risk factors
Breast cancer is the second most commonly diagnosed cancer worldwide and the leading cause of cancer mortality in women. (International Agency for research on cancer, December 2013).
The incidence is quite high in both developed and developing regions as shown in (Fig.3.1)
Fig.3.1 This graph shows the current worldwide Breast Cancer Incidence rates (International Agency for Research on Cancer 2010, Breast Cancer Incidence and Mortality Worldwide).
The number of cancer cases can be described in terms of:
• Incidence, the absolute number of new cases occurring per year.
• Incidence rates, the number of new cases occurring per 100,000 persons per year.
(Global Cancer Statistics, Max Parkin, Paola Pisani, J. Ferlay
, 2008).As indicated in the above shown graph, the distribution of world wide occurring cancer cases is:
• Current World Breast Cancer Incidence rates: 39 per 100,000 population in 2008
•
Current World Breast Cancer Incidence: 1,389,675 new cancer cases diagnosed in 2008More precisely, as the International Agency for Research on Cancer showed, the incidence rates
are ranging from 19.3 in Eastern Africa to 89.7 in Western Europe.However, while the cancer incidence in the developed countries is higher as compared to other continents, the mortality rate is much lower (Fig.3.2).
Fig.3.2
Breast Cancer Incidence and Mortality Worldwide in 2008 (International Agency for Research on Cancer, IARC)We can assume from this data that a higher prevalence of breast cancer in developed countries is counterbalanced by better screening and detection programs as well as readily available “state of the art” treatment protocols. These more developed sanitary programs and higher medical standards most probably lead to relative low mortality rates in more developed regions as compared to less developed countries.
Since the 2008 estimates were published, breast cancer incidence worldwide has increased by more than 20%, while mortality has increased by 14% [1].
These changes over the last few years specifically affected less developed countries, as reported by the IARC:
“Breast cancer is also a leading cause of cancer death in the less developed countries of the world. This is partly because a shift in lifestyle is causing an increase in incidence and partly because clinical advances to combat the disease are not reaching women living in these regions.”
(Dr David Forman, Head of the IARC Section of Cancer Information, Dic. 2013). “The shift towards lifestyles typical of industrialized countries leads to a rising burden of cancers associated with reproductive, dietary, and hormonal risk factors.” [1]To try to understand these “lifestyle’ risk factors that are responsible for the high incidence of breast cancer in the developed countries and are also recently increasing the incidence rate in the less developed ones, the Cancer Research UK tried to summarize the risk factors for breast cancer in the UK, one of the countries with a really high breast cancer incidence (
GLOBOCAN 2012, IARC).Starting from the high proportion of breast cancer cases explained by factors which influence exposure to estrogen, the common parameters used to describe the breast cancer rick factors are:
- reproductive and hormonal factors - obesity
- alcohol
- physical activity
Below a list of analyzed breast cancer risk factors.
Ø Age And Hormonal Status
The older the woman, the higher the ris
k[2].
Women in their 70ies have a risk of 1:8 to develop breast cancer, while a 30 year old women is likely to get breast cancer with a probability of 1:2000 (Fig.3.3).
This is also the result of different events during lifetime that cause a high exposure to oestrogen hormones, like early age at menarche and late menopause. All this factors, together with potential genetic predispositions and the use of hormone replacement therapy to prevent discomforts caused by menopause or in case of prematurely menopause, can increase the breast cancer risk.
The above described events are all linked with the hormonal changes caused by the ovulatory processes. Further endogenous hormones like higher levels of prolactin, insulin, and insulin-like growth factor 1 are positively associated with breast cancer risk
[3] [4] [5] [6] [7].Fig. 3.3
: Estimated risk of developing breast cancer by age, females, UK, 2008 (Cancer Research UK)Ø Reproductive History
One explanation for the increased risk of breast cancer in developed countries is the low number of children per women. Developed countries have fewer children on average and a limited duration of breastfeeding.
Other parameters connected with the effect of hormonal factors are:
- Early age at menarche, lower in developed countries also due to good nutrition.
- Age at first birth: the younger the women is, the lower the risk (in particular for ER+tumors). It is well documented that women in developed countries give birth to their
first child at a relatively late time in their life.
- Low number of children and shorter breastfeeding periods in developed countries.
- Late menopause
[8] [9] [10].
Ø Lifestyle Factors
Bodyweight: Obese pre-menopausal women have a 20% reduction in breast cancer risk, maybe due to the increased likelihood of anovulatory menstrual cycles.
In contrast, in post-menopausal women, obesity increases the risk of developing breast cancer to 30% above the average, because the main endogenous source of estrogen is the conversion of hormones in fatty tissue
[11].Physical activity: 15-20% reduction for the most active women specially post- menopausal, due to the lower levels of estrogen and testosterone
[12].Diet: antioxidants and less consumption of fat intake, especially if saturated, halve the risk of breast cancer
[13].Ø Family History
Hereditary factors contribute around ¼, while environmental factors and lifestyle ¾
[14]. A woman with one affected first degree relative has approximately double the risk of breast cancer.
The risk is higher if the number of affected relatives is more than one and if they have been diagnosed when aged under 50
[14].The highest genetic risk is due to mutations in the breast cancer susceptibility genes BRCA1, BRCA2, with a 45-65% chance of developing the disease by the age of 70 in women carrying this mutation
[15].Other susceptibility genes that if mutated can increase the risk to develop breast cancer are CHEK2, ATM, BRIP1, PALB2, TP53. TP53 mutations have a high-penetrance similar to BRCA ones, but are seen much rarer. The rest of the mentioned genes have an intermediate penetrance
[16].Ø Previous Breast Disease
Ductal carcinoma in situ and lobular carcinoma in situ, are non-invasive conditions of breast cancer.
Women with a previous in situ tumor, have double the risk of invasive breast cancer compared to the general population
[17].The same is true for contralateral breast cancer risk that remains higher for two or more years after a primary unilateral breast cancer, especially if diagnosed before the age of 40 years old
[18].
At the end of this list we can say that breast cancer is often increased in incidence by environmental factors mostly linked with hormone production.
In the past this correlation was affecting mostly developed countries that were and are still facing the increased incidence rates by intensifying screening programs and prevention tests to reduce the breast cancer mortality rate.
During the last years, industrialization and western life style reached less developed countries, causing an increase in numbers of women with breast cancer. Sadly these countries are still facing a lack of early detection programs and access to treatment facilities that causes an increase in breast cancer mortality rates.
3.2 Variety of breast cancer subtypes
The breast is an apocrine gland that produces milk, composed by:
a glandular component, (15-20 lobes), each of which ends into the nipple through a duct;
- fat component, in which the glandular structures grow;
- fibrous support component, which generates subdivisions between the various glandular appendages (Fig. 3.4)
Fig.3.4
Cross-section scheme of the mammary glandFrom different components of the breast, different types of breast cancer can develop, as summarized by the American Cancer Society:
1 Ductual Carcinoma In Situ (Dcis)
It is considered non-invasive or pre-invasive breast cancer. The cells involved are the ones that lined the ducts which after mutations start to highly proliferate.
The difference to invasive cancer is that these cells have not yet spread through the walls of the ducts into the surrounding breast tissue.
About all women diagnosed at this early stage of breast cancer, can be cured.
2 Lobular Carcinoma In Situ (Lcis)
Cells that look like cancer cells are growing in the lobules of the milk-producing glands of the breast, but they do not grow through the wall of the lobules.
The difference to DCSI is that LCIS doesn’t seem to turn invasive, if not treated.
Nevertheless women with LCIS have 7 to 11 fold increased risk of developing invasive cancer in either breast.
3 Invasive Ductual Carcinoma (Idc)
It is the most common type of breast cancer, which originates from a milk duct of the breast and is able to break through the wall of the duct, to grow into the fatty tissue.
At this point it can also spread to other parts of the body through the lymphatic system and bloodstream.
About 8 of 10 invasive breast cancers are infiltrating ductal carcinoma.
4 Invasive Lobular Carcinoma (Ilc)
Starts in the milk-producing glands, lobules and can spread as well to other parts of the body.
Less Common Types:
5 Inflammatory Breast Cancer
Usually there is no single lump or tumor. Inflammatory breast cancer (IBC) makes the skin
on the breast look red, feel warm and sometimes makes it thicker. These changes are caused
This type of breast cancer tends to have a higher chance of spreading and has a worse prognosis than typical invasive ductal or lobular cancer.
6 Triple Negative Breast Cancer
This term is used to describe breast cancers (usually invasive ductal carcinomas) whose cells lack oestrogen receptors and progesterone receptors, and do not have an excess of the HER2 protein on their surfaces. Breast cancers with these characteristics tend to occur more often in younger women and in African-American women. Triple-negative breast cancers tend to grow and spread more quickly than most other types of breast cancer. Chemotherapy is for now the most promising treatment.
7 Pager Didease Of The Nipple
This type of breast cancer starts in the breast ducts and spreads to the skin of the nipple and then to the areola.
Paget disease is almost always associated with either ductal carcinoma in situ (DCIS) or infiltrating ductal carcinoma. Treatment often requires mastectomy. If invasive cancer is present, the prognosis is not as good, and the cancer will need to be staged and treated like any other invasive cancer.
8 Phyllodes Tumor
This very rare breast tumor develops in the stroma (connective tissue) of the breast, in contrast to carcinomas, which develop in the ducts or lobules. These tumors are usually benign but on rare occasions may be malignant.
9 Angiosarcoma
This form of cancer starts in cells that line blood vessels or lymph vessels. It rarely occurs in the breast. When it does, it usually develops as a complication of previous radiation treatments.
Angiosarcoma can also occur in the arms of women who develop lymphedema as a result of lymph node surgery or radiation therapy to treat breast cancer.
10 Special Types Of Invasive Breast Cancer
There are some special types of breast cancer that are sub-types of invasive carcinoma, classified by their microscopical and histological appearance, like the way the cells are arranged within the tumor.
To outline the general process of tumor progression, we can use this schematic description of
tumor progression (Fig.3.5):
Fig. 3.5 Normal breast ducts are composed of the basement membrane and a layer of luminal epithelial and myoepithelial cells. Cells composing the stroma include various leukocytes, fibroblasts, myofibroblasts, and endothelial cells. In in situ carcinomas the myoepithelial cells are epigenetically and phenotypically altered and their number decreases potentially due to degradation of the basement membrane. At the same time, the number of stromal fibroblasts, myofibroblasts, lymphocytes, and endothelial cells increases. Loss of myoepithelial cells and basement membrane results in invasive carcinomas, in which tumor cells can invade surrounding tissues and can migrate to distant organs, eventually leading to metastases [19].
Apart from the nature of the lesions present in epithelial cancer cells, an important role in the cancer progression is played by the cells composing the microenvironment. For instance stromal cells have been found to harbor similar epigenetic changes to the ones present in cancer cells.
3.3 Breast Cancer treatments currently used in clinic
There are numerous breast cancer treatments currently used in the clinic, which are tailored towards the tumor subtype and genetic lesion:
1. Surgery, to remove the tumor with a margin of normal breast tissue, called Partial Mastectomy.
In case of an invasive malignant tumor, a Total Mastectomy is necessary, to remove the whole breast, sometimes with associated lymph nodes, in order to check the eventual presence of metastasis.
An even more drastic measure is a Radical Mastectomy, that also removes the lining over chest muscles and sometimes part of the chest wall muscles.
Often, to decrease the size of the tumor before the surgery, chemotherapy, called neoadjuvant therapy, is given.
After the surgery, radiation therapy, chemotherapy, or hormone therapy can be performed to kill any cancer cells eventually left, in order to lower the risk of relapses. This is called adjuvant therapy.
2. Radiation therapy is related with the use of high energy x-rays or other types of radiation to kill cancer cells or keep them from growing.
The first type of radiation therapy is external, through a machine that sends radiation toward the cancer.
The second type is internal and uses radioactive substances sealed in needles, seeds, wires or
catheters, placed directly into or near the cancer.
3. Chemotherapy involves the use of drugs to stop the growth of cancer cells killing them or stopping them from dividing. The drugs can be administered in the bloodstream or directly in the areas of the tumor.
4. Hormone therapy has the goal to block hormones production or action in order to stop cancer cells from growing. Some hormones, such as estrogen, can help certain cancers to grow.
The ovaries mainly produce estrogen, therefore ovarian ablation can be performed to stop the estrogen production.
Hormone therapy with Tamoxifen is often given to patients with early stages of breast cancer or metastatic ones ER+ (estrogen receptor positive). Tamoxifen is an antagonist of the estrogen, that binds to the estrogen receptor but doesn’t activate it. In this way the estrogen signal is silenced.
Another hormone therapy uses the aromatase inhibitor, which blocks the enzyme aromatase from converting androgen into estrogen.
5. Targeted therapy uses drugs or other substances to identify and attack specific cancer cells without harming normal cells. Monoclonal antibody and tyrosine kinase inhibitors are two examples of substance classes that are used in targeted therapies.
Monoclonal antibodies can identify specifically the mutated form of proteins on cancer cells which help cancer cells to grow. The antibodies bind the protein (e.g. a mutated transmembrane receptor) and kill the cancer cells by silencing the activity of the bound protein. This way they block tumor cell proliferation, or keep them from spreading. These specific antibodies can be used alone or as carrier for drugs, toxins, or radioactive substances driven directly to cancer cells.
One example is Trastuzumab a Her2-targeted humanized monoclonal antibody that binds to the subdomain IV of the Her2 receptor and disrupts ligand independent Her2 dimerization. This causes the block of the MAPK pathway, which is partly responsible for cell proliferation, differentiation, migration and angiogenesis.
An other example for a monoclonal antibody is Pertuzumab, an anti-HER2 humanized antibody that binds to the subdomain II of HER2 and blocks ligand activating pairing with other Her receptor including Her3, one of the only receptors that can directly activate the PI3K pathway to drive cell survival signaling. Therefore Pertuzumab is able to block the PI3K and the MAPK pathway, and automatically the tumor growth.
Both antibodies, often used in combination for the treatment of HER2+ tumor, are also able to recruit immune cells which are stimulated to attack and destroy tumor cells in a process called antibody dependent cellular cytotoxicity (ADCC).
Tyrosine kinase inhibitors are targeted therapy drugs that block signals needed for tumor growth.
A prominent inhibitor in breast cancer available for clinical uses is Lapatinib, an inhibitor of the intracellular tyrosine kinase domains of both Epidermal Growth Factor Receptor (EGFR
[ErbB1]) and of Human Epidermal Receptor Type 2 (HER-2 [ErbB2]) receptors. Lapatinib is used for the treatment of patients with advanced or metastatic breast cancer whose tumors
overexpress HER2 and who have received prior therapy including an anthracycline, a taxane, and trastuzumab [20]
.3.4 Oncogenes and tumor suppressors, “drivers” for Breast Cancer Breast cancer can be divided in five distinct molecular subtypes:
- Basal like - Luminal A - Luminal B - HER2+/ER
- Normal breast-like
This grouping exists mostly as research settings, to investigate if the molecular differences among the groups could help in planning treatment or develop new therapies.
The Luminal A is the most frequent, with a prevalence of 40%. It tends to be ER+ and/or PR+, HER2-, with high survival rates and low recurrence rates, often treated with hormone therapy.
The tumor cells in this case look like the cells of breast cancer that start in the inner, luminal, cells lining the mammary ducts.
The Luminal B has a prevalence of 20% and is ER+ and/or PR+, HER2+ with a high number of cancer cells actively dividing (highly positive for ki67). P53 mutation is present in higher rates compared with the Luminal A, and the survival rates is fairly high.
The basal-like/triple negative breast cancer types, are often associated. They are ER-, PR-, HER2-, and for this reason it is not possible to give any hormone or targeted therapy. Often treated with surgery, radiation and chemotherapy, this subtype is much faster and invasive than all the rest. The cells look like those of the outer, basal, cells surrounding the mammary ducts.
Most of them contain p53 mutations. The prevalence is about 15/20%, and often also present BRCA1 mutation.
The HER2 type is mostly Her2+ and only a 30% Her2-. It is ER-, PR- and often contains p53 mutations. It has a fairly poor prognosis and frequent recurrence and metastases.
The last classified subtype is the Normal-like case that has a prevalence of 10% and do not present any particular molecular composition. These tumors are usually small and tent to have good prognosis
.The origin of breast cancer differentiation in molecular subtypes is still not known, but a hypothetical model supposes the dependence with the different stages of commitment of the cell of origin (Fig. 3.6):
Fig.3.6 Hypothetical models explaining breast tumor subtypes. Based on the cell of origin hypothesis (A), each tumor subtype is initiated in a different cell type (presumably stem or progenitor cell), whereas according to the model depicted in (B), the cell of origin can be the same for different tumor subtypes and the tumor phenotype is primarily determined by acquired genetic and epigenetic events [21].
The malignancy of cancer cells is seen to be a stepwise process that involves genetic and
epigenetic alterations in which also cells of the microenvironment are involved, such as immune,
vascular and stromal cells.
Mutations in genes that that drive cancer development and progression can be somatic, occurring during the patient’s lifetime, or inherited. Besides the genetic alteration that causes cancer development, also a series of genetic polymorphisms can be inherited and can cause a predisposition to develop cancer.
In terms of genes directly responsible for tumor growth, they can be classified in two big groups, Oncogenes and Tumorsuppressors.
The oncogenes directly drive the tumor growth by increasing the proliferation rate of the cells.
The mutation in the proto-oncogenes, the wild type version of the oncogenes, causes an overexpression of the gene or the acquisition of an enhanced function for the gene protein product. The mutation can be a point mutation, a chromosomal translocation, or a fusion with other genes that causes an enhanced function, and cooperation with other genetic or epigenetic changes.
One example of this group is HER-2, the gene for the human epithelial receptor 2, a transmembranal tyrosine kinase growth factor receptor that leads to multiple transduction cascades acting through a variety of pathways such as MAPK and PI3K, responsible for cell proliferation, angiogenesis, migration and survival. If this oncogene is mutated and overexpressed, all these pathways are constitutively activated causing an uncontrolled cell growth.
Another example for an oncogene is c-Myc, often overexpressed in breast cancer, that encode a nuclear phosphoprotein that acts as a transcriptional regulator involved in cellular proliferation, differentiation and apoptosis.
Regarding tumor suppressor genes, they usually negatively regulate cell growth and proliferation.
In case of mutations they lose this regulatory function and consequently the broken brake for cellular proliferation and growth causes tumor growth. The cause of dysfunction of a tumor suppressor can also be epigenetic, such as methylation of the promoter, or can be due to increased proteasome degradation, or abnormalities in partner proteins interacting with the tumor suppressor gene product.
One example of a tumorsuppressor is p53, a gene that encodes for a protein with multiple functions. Its main role is to control the health of the cell by regulating the cell cycle and by triggering apoptosis or differentiation. In general it is a multifunctional protein, for this reason called “the guardian of the genome”, and mutations in different domains may have different and specific consequences.
Other tumor suppressors are BRCA-1 and BRCA-2, proteins involved in DNA repair mechanisms, and if mutated cause the accumulation of mutations in the entire genome
[22].The oncogenic or tumorsuppressor lesions can appear in different cells, in terms of type and stage of commitment.
For this reason different models have been postulated to try to correlate different types of breast cancer with different cells of origin, in order to clarify the tumorigenic process and develop new therapies (Fig. 3.6). Nevertheless the key points of breast cancer progression are still not known and under investigation.
3.5 Targeted therapy and oncogene dependence
A normal cell became a cancer cells by accumulating mutations in multiple genes that cause changes in their expression and more in general chromosomal alterations.
As above described, the two main genetic/epigenetic processes behind tumor growth, involve the acquisition of activating mutations in oncogenes (like Her2 or Myc) and inactivation of tumor suppressors (like p53, BRACA, p10).
Commonly, among different cancer types, there is the dependence of the cancer cells on the
expression of the primary oncogene. This principle is called Oncogene addiction, according to
which tumor cells are dependent on a single oncogenic activity for their survival and proliferation.
Shutting down the oncogene expression, the tumor cells undergo apoptotis and the tumor mass drastically regresses.
This is due to a physiologically dependence on a continued activity of specific activated or overexpressed oncogenes for maintenance of the malignant phenotype [23].
This phenomena applies not only for the oncogenes, but also for the tumorsuppressors. Indeed providing mutated cells with the functional wild type product, the results are inhibition of tumor
growth and induction of apoptosis [24].What is interestingly underlined by I. Bernard Weinstein in his Science review, is that the correction of just one mutation is enough to block tumor growth.
This oncogene dependence and “hypersensitivity” to the process of restoring one single suppressor gene expression, may be due to the strict homeostasis between positive and negative acting factors present in tumor cells. This homeostasis must be maintained to ensure the structural and functional integrity of cancer cells, and can be considered its Achilles heel [25].
This weakness is the foundation on which the principle of targeted therapy is based.
A deeper investigation on the roles of different oncogenes in different tumor samples, will be fundamental for the synthesis of specific molecules able to recognize tumor cells and attack them specifically [26] [27].
A lot has been done in these last few years, from the discovery of the receptor tyrosine kinase, revealed afterwards as a perfect target for cancer therapy [28] [29] to the synthesis of tyrosine
kinase inhibitors. Deeper investigations are of course needed, due to the difficulty to synthetizespecific molecules that exclusively target the mutated macromolecules crucial for cancer cells, so they do not harm normal tissues [30].
Nevertheless important steps have been done such as the synthesis of molecules already above cited like Trastuzumab, also called Herceptin. This monoclonal antibody binds to the extracellular domain of HER2 and blocks its dimerization and consequently the downstream pathway associated.
Another molecule used for targeted therapy is Lapatinib, an ATP-competitive tyrosine kinase inhibitor that dually the human epidermal growth factor receptors 1 and 2 (HER2). It enters the cell and binds to the intracellular domain of the tyrosine kinase receptor, allowing for complete blockage of the autophosphorylation reaction and a complete halt to the downstream cascade of events. (A complete list of molecules now available for treatment is published in the National Cancer Institute at the National Institutes Heath [30] [31]).
The behavior of tumors on targeted therapy and subsequent relapse is not only dependent on the nature of the oncogene, but also on the tissue context.
This can be exemplified by the role of Myc in sarcomas versus mammary epithelial tumors: As described by Jain M et al. [32], mouse osteogenic sarcomas induced by the overexpression of Myc under a tetracycline inducible system, react with tumor regression upon silencing of MYC, as expected. However, surprisingly, re-indunction of Myc expression is not followed by the regrowth of the sarcoma as expected, but it induces apoptosis .
In a different cellular environment, like the mammary gland, the Myc effect is different. D'Cruz et al. [33], described the effects of the Myc overexpression in mammary glands, able to promote tumor growth. The result of the de-induction is a regression of the tumor only in 30% of the cases, while the rest acquire Myc-indipendent secondary survival pathway activation, via mutation in K- ras gene, that maintain tumor growth.
This means that different mutated genes have different roles in different cell types, increasing the problems in finding cancer therapy. Therefore, what is primarily important, is to investigate the eventual different oncogenes effects in different cellular context, in order to better understand the specific tumor progression mechanisms (
Felsher Journal Compilation 2008).
Deeper investigations about targeted therapies are also necessary to have more effective
inhibitors even against known secondary mutations that arise during primary treatments. One is
the frequent insurgence of drug resistance which causes the failure of the therapy [34] [35]. To
enhance the efficacy of treatment synthetic lethal approaches are designed. The idea being to
combine targeted therapy with sublethal doses of treatments targeting more general cellular pathways. Non oncogenic pathways, like DNA damage, replication stress, metabolic and oxidative stress, angiogenesis, hypoxia and stromal support, are under investigation as likely alternative additions to targeted therapy [36].
It will be imperative to overcome the incapacity to prevent tumor relapses. Breast cancer relapses, by far are the most resistant and aggressive forms of breast cancer, are still not curable and cause the high world wide mortality rates.
3.6 Targeted therapy in clinical use: the problem of resistance and relapses Clinicians currently face the problem of drug resistance related with the use of targeted therapy [37] and a high rate of mortality related with breast cancer relapses. The frequency of relapses is depending on the breast cancer subtype [38]
.As previously mentioned, the most widely used targeted therapies are Trastuzumab, Pertuzumab and Lapatinib. Nevertheless, a significant number of patients with Her2+ breast cancer, treated with these drugs, often relapse.
These recurrences are often driven by the acquisition of different, additional Her2 alterations, which allow the tumor cells to escape the initial Her2 inhibition. Alternatively, the Her2 silencing can be bypassed by other pathways that activate similar effector molecules downstream
[39].Different possible mechanism of relapses have been suggested, among them incomplete regression that leaves a small number of transformed cells, able to repopulate the tumor. This hypothesis has been investigated by looking at the resistance mechanisms acquired in patients with lung or gastrointestinal cancer. Surviving cells displayed the acquisition of “gatekeeper”
kinase domain mutations, which proved to be responsible for drug resistance.
In the case of Her2 resistant breast cancer relapse, a potential mutation in the juxtamembrane region of Her2 that contains the binding epitope of Trastuzumab, could be responsible for primary drug resistance. Another observed possibility for a resistance mechanism is the splicing variant that eliminates exon 16 in the extracellular domain of the HER2 receptor, to stabilize HER2 homodimers and potentially prevent their disruption upon antibody binding. Nevertheless , since this specific splicing isoform interacts and directly activates the Src tyrosine kinase survival pathway, an inhibitor for Src, (dasatinib) can be employed.
The most frequent somatic alterations found, are gain of function mutations in PI3K/AKT as well as alterations of the normal apoptotic machinery, deregulation of cell cycle controls and the immunomodulatory factors, often stimulated by the administration of Trastuzumab.
Of course implication of other co-family members of Her2, that could substitute the lack of HER2, and that are not targeted by Trastuzumab, or cross-talk with other receptors outside of ErbB family, have been found.
The last hypothesis is the possibility of the cellular overexpression of molecules able to bind Trastuzumab, and abrogate its function.
Most likely drug resistance and tumor relapse have similar action mechanisms.
3.7 Transgenic mouse models to study oncogene dependence
To study oncogene dependence in breast cancer, it is necessary to develop animal models that can recapitulate the human tumor situation..
Mouse models have proven to be very useful to recapitulate different human diseases and are able to reproduce the progression of human breast cancer [40]
.To study at the same time breast tumor progression and oncogene dependence, a doxycycline-
inducible system has been used.
The first transgenic mouse model contained the mouse mammary tumor virus long terminal repeat, MMTV-LTR, used as promoter of the Tet-responsive transactivator tTA that is specifically expressed in the mammary glands. In this way tTA is constitutively able to activate the expression of a second transgene by binding the Tet operator promoter region to drive expression of the respective transgene.
This system is called Tet-Off inducible system, but has evident limits, like the observed possibility to have small fractions of mammary epithelial cells still expressing the transgene after the doxycycline administration.
A mouse model with a homogeneous expression of the transgenes in all the mammary gland cells is a relevant approach to study breast cancer development. The tet-on system, that I am currently using for my work, also offers the possibility to have a strictly regulation of transgenes, with rapid kinetics of induction and de-induction of the oncogenes expression.
The Tet-On system, based on a reverse tetracycline-controlled transactivator, rtTA, relies on a fusion protein comprised of the TetR repressor and the VP16 transactivation domain. The difference to tTA is a four amino acid change in the tetR DNA binding moiety that alters rtTA’s binding property in a way that can only recognize the tetO sequences in the TRE of the target transgene in the presence of the doxycycline effector. Using different reporter genes it has shown homogeneous spatially transgene expression in the mammary epithelium of transgenic mice treated with dox. This expression is absent in wild type cells or in transgenic ones without administration of doxycycline. This expression is specific and rapidly interrupted with the dox withdrawal [41].
Starting from the basis of this model, different transgenic mice for different transgenes have been generated.
For example, often involved in breast cancer growth is the oncogene
Myc [42] [43].The c-Myc inducible system in mammary glands (under the control of MMTV-LTR rtTA)has been described by D’Cruz et al. [33]. Human c-Myc is selectively expressed in the mammary and in the salivary gland cells, after administering doxycycline to the mice through the food.
Morphological analysis after 30 days of doxycycline, showed hyperplastic mammary glands, but at the same time an increased number of apoptotic cells.
Induction with doxycycline for 4 months displayed more severe morphological abnormalities.
Interestingly, mice that were fed for 30 weeks with dox and than for 12 weeks without dox, had histologically normal mammary glands, indicating that the hyperplastic lesions were linked to the overexpression of c-Myc. In this work has also been observed that after an average of 14 days from the dox withdrawal, in contrast to the disappearance of invasive mammary adenocarcinomas, another consistent subset of tumors continued to grow.
After confirming the absence of exogenous c-Myc expression and endogenous one, the analysis showed a selected spontaneous activation of the Ras pathway in the analyzed cells. This mutation is able to drive the progression of the tumor to a stage that is no longer depended on c-Myc for its growth and survival.
Breast tumor progression is linked to the timing of the normal breast development, and for this reason it is important to study the impact of an oncogene in different developmental stages, to better understand is function in normal and tumorigenic cells as shown in a detailed research of
Collin M.Blakely et al. [44], concluding thatMYC-induced mammary tumorigenesis may be similarly affected by the mammary developmental stage at which this oncogene is expressed.
Important observations have also been done by Robert B. Boxer et al [45]. After mammary gland
tumor growth induced by Myc overexpression, the dox withdrawal caused the full regression of a
minority of tumors, while the rest, were only partially regressed. Indeed these partially regressed
tumors rapidly reinitiated their growth, indicating that a large number of Myc induced tumors
have acquired the ability to grow in absence of the oncogene overexpression. Following the mice
with totally regressed tumors, interestingly after an average of 80 days they developed a relapse
without the presence of dox. This means that even fully regressed mammary glands harbor
residual neoplastic cells ready to reacquire their full malignant potential.
A second system important for my line of research, is the Her2 inducible one
[46].The breast cancer mouse model has been described by Susan E. Moody et al. [47]. In this case a constitutively active version of the Her2 receptor tyrosine kinase, due to the point mutation in its transmembrane domain, has been cloned downstream of the minimal Tet operator.
After four days of doxycycline, hyperplastic abnormalities have been found in the mammary ductal trees. Chronic induction resulted in multiple mammary tumors.
After 1 day of dox withdrawal, the expression of Her2 was rapidly downregulated, and the vast majority of tumors, completely regressed. This means that most of the tumorigenic cells remain dependent on Her2 for the maintenance of their malignant state. Analyses of the regressing tumors, showed a decrease in proliferation and increase in apoptosis.
Nevertheless, in this case, associated with breast cancer, also lung metastasis were detected that showed a dependence on Her2 activation for their transformed state.
In 7 mice a total number of 8 relapses have been found, meaning that a subpopulation of neoplastic mammary epithelial cells are able to escape their requirement of Her2 to reestablish a malignant phenotype, but this aspect is still under investigation
[48].The recurrence of relapses is the worst prognosis, often followed by death. For this reason it is very important to understand the downstream consequence of Her2 over activation.
What is interesting to mention is that, primary human breast cancers present an overexpression of the wild type ERBB2, due to gene amplification and presence of alternative splicing forms of constitutively activated the Her2 protein.
The mouse model that overexpress Her2, develops mammary gland tumors, and in these tumors have been often found spontaneous activating mutations in the Her2 gene sequence. This means that mammary tumors seem to have a strong selective pressure for the occurrence of activating mutations, and in mice this recapitulates the effects of the alternative splicing forms found in human [49].
The potential of this system is not only to recapitulate the human breast tumor progression, but also the first steps of the clinical treatment with targeted therapy, in which the treatment with HER2 inhibitors is obtained by the dox withdrawal, that switch off the overexpression of the oncogene.
In my case the mouse model used is a combination of these two above presented models, with the presence of both sequences of the oncogenes Myc and Her2, in a tri-transgenic mouse TetOMyc/TetONeu(Her2)/MMTVrtTA.
In human breast cancer this combination of Myc and Her2 has been found in 2,4% of total cases and is often related with a poor prognosis [50] [51] [52].
The characteristics of this model are still under investigation and will be also partly described in the Results and Discussion paragraphs.
3.8 Oncogenes chosen for the mouse model: Her2 and Myc
The first oncogene, Her2, as mentioned before, encodes for the human epithelial receptor 2, also known as Neu or erbB-2. The gene is localized on chromosome 17q and the protein encoded is a 185-kDa transmembrane tyrosine kinase growth factor receptor.
It has an extracellular domain, a transmembrane domain that includes two cysteine-rich repeat clusters and an intracellular kinase domain.
Her2, differs from the other Her family member receptors, has no known ligand and it exists in an
open conformation, continually available for homo or hetero-dimerization with other Her
receptors. The dimerization is followed by the phosphorylation of several tyrosines in the
receptor’s C-termini.
The intracellular domain serves as a docking sites for a number of Src homology (SH)2- containing adaptors and signal transducers that mediate the transforming effects of this receptor network (Fig. 3.7).
After dimerization and phosphorylation, multiple transduction cascades are activated and involve pathways like MAP kinase, 3-kinase/AKT pathways, which end in different intracellular signals as proliferation, angiogenesis, altered cell-cell interactions, increased cell motility, resistance to apoptosis
[53].The most potent oncogenic receptor pair is the Her2-Her3 one.
HER-2 is amplified and overexpressed in 20%-30% of invasive breast with a range of 3 to 42 gene copies number. The gene is often amplified and only rarely rearranged.
The Her2 gene sequence used for our tri-transgenic mice is the rat one.
Fig.3.7 EGFR pathway.
The second oncogene used in our studies is Myc, which is localized on chromosome 8q24 and encodes a nuclear phosphoprotein that acts as a transcriptional regulator involved in cellular proliferation, differentiation and apoptosis (Fig.3.8) [53].
The frequency of Myc amplification is 20% with a range o 3 to 14 gene copies. In human breast cancer c-Myc has always been found amplified and a rearrangement was detected in only one infiltrating cancer [54] [55].
The c-Myc gene used for our tri-transgenic mice is the human c-Myc sequence.
Fig.3.8 Myc pathway
.
3.9 In vitro complementary approach
Investigating tumor growth in vivo is a slow process and doesn’t allow to follow one single tumorigenic structure during its changes.
For this reason is is important to use an in vitro system that can recapitulate the in vivo tumorigenesis by reproducing the natural microenvironment.
With this aim, the three-dimensional (3D) cell culture method has been adapted to study primary mouse mammary transgenic epithelial cells [56].
These mammary epithelial cells come from the previously mentioned tri-transgenic mouse TetOMyc/TetONeu(Her2)/MMTVrtTA.
The importance of this system is the capacity to recapitulate the organotypic growth of the mammary gland acini with a polarized phenotype, specialized cell–cell contacts, and attachment to an underlying basement membrane, fundamental characteristic, especially for epithelial cells (Fig. 3.9).
Fig. 3.9 Scheme of a section of a glandular acinus.
The mammary gland acini are composed by a bi-layer of luminal epithelial cells and myoepithelial cells, surrounded by basement membrane and stromal tissue. The luminal epithelial cells are the functionally active milk-producing cells, most likely target cells for carcinogenesis.
The myoepithelial cells are the contractile portion of the gland, that, surrounding the luminal cells causes the milk secretion.
This means that for most of the surface, the myoepithelial cells are juxtaposed between the luminal epithelial cells and the surrounding basement membrane.
Therefore, the myoepithelial cells are not only situated in an ideal position to communicate between these two compartments, but they can also provide important regulatory signals for the maintenance of normal breast structure.
The myoephitelial cells are attached to the basement membrane by hemidesmosomes and to the adjacent luminal epithelial and myoephitelial cells by desmosomes.
The myoepithelial cells have a further important role, due to the secretion of many basement membrane proteins, such as laminins. This is foundamental for the polarization of the luminal epithelial acini [57].
Other myoephitelial secreted molecules, like desmosomal cadherins, are also important for the
polarity structure of the acinus, and ablation of these molecules, together with the laminin, cause
lack of apico-basolateral polarity of the epithelial cells [58].
Indeed, the epithelial cells of the mammary gland have an apical and a basal polarity (Fig. 3.10).
Fig.3.10 Different localization of subcellular molecules in polarized epithelial cells.
The apical side is the one that look into the hallow lumen and the signals that come to this side of the cell are quite different from the ones that come from the basal side. The basal side of the cells, in fact, is the one that is in direct contact with the myoephitelial cells. In some regions, the luminal epithelial cells are directly in contact with the extracellular matrix (ECM), but often, the overlying layer of myoepithelial cells mediates the communication with the ECM.
To be ready to receive specific types of signals from the ECM, the membrane and the cytoplasm organization and composition are strongly different in this basal side of the luminal epithelial cells. This is the reason, why we talk about polarity.
The polarity is fundamental for their functionality and is guaranteed only by the tri-dimensional structure of the acini and the different signals coming from the two sides, the apical and the basal one.
The only way to preserve this structural/functional organization is to allow the single mammary gland cells to grow and for 3d acini in a 3d cells culture.
The organized structure of the normal mammary gland is completely lost in the tumor samples.
What is known is that in some tumor types myoepithelial cells rarely transformed and during breast cancer progression are outnumbered by cancer cells and gradually disappear. This is consisten with the hypotesis that myoepithelial cells are tumor suppressor. Often a consequence of the tumor progression, the lost of consistence of the myoepithelial layer could be [59]. If their decreasing in number is a consequence of the tumor progression or is one of the cause of the tumor growth is not yeat clear, but for sure the myoephitelial cells are directly involved in the preservetion of the tissue integrity and consequently in its lost during tumorigenic processes [60].
3.10 Survival and polarity pathways: possible targets to reduce the frequency of relapses
As described in the previous paragraphs, the main three-dimensional structure of the mammary gland acini is responsible to the survival and functionality of the gland. This is due to the dependence of the epithelial cells to polarized signals coming from different sides of the acinar structure, in order to maintain their functionality.
As partly shown in the Fig.3.11, the presence of an apical side of the acinus that look into the
hollow lumen, and a basal side, that is in direct contact with the myoepithelial cells and the
basement membrane, causes the structural differentiation of the subcellular component of the
epithelial cells. Each side presents different composition in terms of molecules, due to the differences in the type of signals received.
The strongest difference is due to the presence of huge stimulations coming to the basal side, from the basal membrane. The membrane, in fact, contains important molecules that, binding to epithelial cell receptors drive the activation of the most important intracellular pathway for cell survival and proliferation.
Due to these signals and to the mechanical constrictions caused by the presence of myoepithelial cells, the luminal epithelial cells are forced to maintain a certain inter and intra cellular structural organization.
A mentioned before, one of the reasons for the still high mortality rates of breast cancer, is the recurrence of relapses after a successful treatment of the primary tumor.
The final aim of my investigation is to try to decrease the possibility of relapses recurrence by targeting and reducing the number of luminal epithelial cells that survived the dox withdrawal.
These cells, in fact, as mentioned in the previous paragraphs and explained more in detail in the Results section, are tumorigenic cells that after dox withdrawal regress to a apparent normal polarized epithelial cell layer. However, after a certain period, these cells are able to form relapses without the administration of dox.
To reach this goal, we focused to one of the most important characteristic of the epithelial cells, the polarity. We thought that blocking important pathways for the cellular survival and for the polarity stability, we could have the chance to push these cells to an apoptotic fate.
Understanding the process of epithelial polarization, in fact, helps to understand the progression of carcinomas [61]
.It has also been shown that the loss of epithelial cell polarity may have an important role in both initiation of tumourigenesis and in later stages of tumor development, favoring the progression of benign tumors from to a malignancy state [62]
.The epithelial morphogenesis in vitro is called Cystogenesis. Cysts are spherical monolayers of epithelial cells enclosing a central lumen, in which the cells are connected by specialized junctions and cell-cell adhesion structures lying in the baso-lateral sides such as E-cadherin, while the apical side is the one facing the lumen with tight junctions such as ZO1.
Fig.3.11 Tight junctions and adherence junctions in mammary gland luminal epithelial cells.
Basal membrane Apical membrane
Fig.3.12 Integrin receptors and extracellular matrix.
The interaction between cells and various components of the extracellular matrix (ECM) is of vital importance for a number of cellular functions such as cell survival and death (apoptosis), proliferation, differentiation, cell shape modulation, actin organization, migration, and gene expression [63].
Indeed, epithelial cells are physically connected to the ECM, via integrin receptors. The integrin family of transmembrane receptors links the extracellular matrix (ECM) to the intracellular actin cytoskeleton at points of cell–substratum interaction termed focal adhesions (Fig.3.12). Integrin clustering can initiate intracellular signaling events that promote cell proliferation, survival and migration in both normal and tumorigenic cell contexts (Fig.3.13).
Integrins are trans heterodimers with α and β subunit, and the ones expressed in the epithelium include β1 integrin that dimerizes with different α subunit such as α6β4, α5β5, and α5β6.
α 1 β 1 and α 2 β 1 recognize collagen receptors and α 3 β 1 laminin isoforms.
Some of these integrins can also be found in the baso-lateral surface.
The signals coming to the cells from the basal membrane through the integrins receptors are the activation of important survival pathways and cell-cell adhesion signals, that drive the formation of focal adhesions and a stabile structure of polarized acini.
Integrins can, therefore, be activated in two ways: either as a result of their detection of changes in the microenvironment of the cell (outside-in signaling) or in response to signals that originate from the inner compartment of the cell (inside-out signaling) [64] [65] [66] [67] [68]. In a molecular level, most of the integrin-associated molecules in focal adhesions are multifunctional.
They associate integrins and actin cytoskeleton and serve as scaffolds for the attachment of enzymes such as kinases and phosphatases that modify and regulate these complex interactions.
Currently, more than 60 integrin-associated proteins including talin, actinin, filamin, paxillin, focal adhesion kinase (FAK), integrin linked kinase (ILK), PINCH, and Parvins have been identified.
The ‘‘outside-in signaling’’ of integrins begins with the binding of the ECM ligands to integrins,
followed by clustering of integrins and the recruitment of actin filaments and signaling
complexes to the cytoplasmic domain of integrins [69]. These initial complexes, which in
cultured cells are often referred to as ‘‘focal complexes’’ will, in turn, give rise to mature
structures that consist of larger and more complicated protein assemblies. One example is the
integrin signaling through extracellular signal regulated kinase (Erk), the principal mitogen
activated protein kinase (MAP kinase) that also occurs during cell adhesion.
Fig3.13 Highlights of some important integrin signaling pathways in epithelial cells.
Going through the integrin-associated proteins mentioned before, FAK is an interesting one, involved in different pathways (Fig.3.14).
Its first activity is kinase dependent and is performed mostly by the Src family kinase.
Src is the first discovered protein to have tyrosine kinase activity and if overexpressed it plays a role of oncogene [70]. It can act as an upstream or downstream modulator of several receptor molecules, as well as non-receptor tyrosine kinases, like FAK. It has been reported that in particular for breast cancer, there is an enhanced expression and activity of c-Src kinase that may play a role in carcinogenesis, or in maintenance of the malignant phenotype [71]. If phosphorylated, Src can associate with actin filaments that drive Scr to cell-cell and cell-matrix adhesion sites, from where the major transduction events start. Examples are receptor tyrosine kinases which mainly affects cells growth, proliferation, migration and adhesion receptors, including integrins and E-cadherin that regulate cytoskeletal functions [71].
FAK has also a kinase-independent activity, an antiapopotic function played in the nucleus. In the nucleus it also regulates the gene expression, by controlling the chromatin structure. Even though only the N-terminal fragment contains a nuclear localization sequences, both full length and C- terminus have been found in the nucleus.
A further among the very numerous functions of FAK, is the impairment loss of E-cadherin from the cell surface, consistent with the role of FAK in regulating the disassembly of E-cadherin- based junctions.
FAK is primarily recruited to sites of integrin clustering via interactions between its C-terminal domain and integrin-associated proteins such as talin and paxillin. The cytoplasmic tail of b- integrins (b1, b3 and b5) facilitates FAK activation through undefined mechanism(s), potentially involving FAK clustering, autophosphorylation at Y397 and a mechanical linkage of integrins to the actin cytoskeleton.
RNAi studies showed that FAK expression promotes cell polarization and that Y397 FAK phosphorylation plays an important but so far undefined role in the spatial organization of the leading edge in migrating cells. The inhibition of the phosphorylation in Y397 due to a point mutation Y-F, has been shown to inhibit FAK to ERK/MAP kinase signaling linkage and this is one pathway through which FAK promote cell proliferation. Additionally, it is known that FAK can promote cell survival both by enhancing phosphatidylinositol 3-kinase (PI3K)- mediated activation of AKT and by suppressing p53- mediated apoptosis in a PI3K-independent manner (FERM domain can become localized to the nucleus and may directly bind to the transactivation region of p53 to limit p53’s transcriptional activity)
One of the most known characteristics of cancer cells is their ability to proliferate regardless the
adhesion to a substrate. In the past FAK overexpression have been linked to the acquisition of a
malignant status for the cell, the resistance to anoikis, a programmed cell death induced by anchorage-dependent cells, detaching from the surrounding ECM [72]. The up regulation of FAK seems to end in the acquired ability of the cells to grow in an adhesion-independent manner. We can conclude that FAK, a non-receptor tyrosine kinase, that transduces signals from integrin receptors and enhances signaling of many receptor tyrosine kinase like EGFR, Erb family and angiogenesis receptors, is a key molecule involved in the control of cell proliferation, migration, morphology and survival [73].
Fig.3.14 Structural model of full-‐length FAK (FERM, green; kinase, cyan; FAT, magenta)
Another important molecule for cell polarity and lumen formation in glandular epithelium is the
Integrin linked kinase, ILK (Fig.3.15). It plays a role as multifunctional adaptor protein linkingfocal adhesions to the actin cytoskeleton. ILK binds directly to b1 and b3 integrin subunits.
Fig.3.15 Structural model of ILK
Its role is directly linked to the one of β1 integrin, the ablation of which results in loss of
polarized cysts [74]. In particular deletion of β1 integrin resulted in displacement of ILK from the
basal cell surface and dephosphorilation of FAK.
ILK can phosphorylate several proteins, including protein kinase B PKB/Akt, Gsk-3, and myosin light chain. The activation of ILK, either by integrin clustering or by growth factors, involves multiple cell signaling pathways that regulate cell survival, proliferation, and differentiation.
ILK has also been shown as regulator for tumor angiogenesis by regulating MAPK activation through the stimulation of the expression of VEGF [75]. ILK overexpression promotes anchorage-independent cell growth and cell cycle progression [76] [77] [78] [79]. ILK overexpression in epithelial cells leads to the down-regulation of E-cadherin and disruption of cell–cell adhesions [76], as well as inhibition of suspension induced apoptosis (anoikis) [80] [81]
[82] [84] [85] [86] [87]. Modest overexpression of ILK in intestinal and mammary epithelial cells promotes cell invasion and is accompanied by translocation of catenin to the nucleus, formation of the complex between catenin and the LEF1 transcription factor, and subsequent transcriptional activation of pro-survival genes such as MMP-9, cyclins, and c-MYC [88] [89] [90]. Finally, clinic studies have shown that ILK expression level increases in a number of malignant tumors, and its expression levels are often correlated with the tumor grade.
Fig.3.16 General intracellular pathways starting from Integrin receptors.
Inhibition of ILK kinase activity, on the other hand, suppresses cell growth in culture as well as growth of human colon carcinoma cells in SCID mice [91]. Overexpression of ILK in mammary glands of transgenic mice leads to tumor formation [78]. Similarly, pharmacological inhibition of ILK in prostate carcinoma cells induces a less rapid proliferation [92]. Furthermore, it has been shown that stable transfected intestinal and mammary epithelial cells over- expressing ILK adopt a highly invasive phenotype, which ultimately can lead to tumor formation [93]. Constitutive activation of integrin-dependent signaling events by oncogenes provides a molecular explanation for the link between growth and adhesion [94]. Understanding the importance of these two molecules ILK and FAK, and their published involvement in malignant cellular phenotype and tumor growth when overexpressed, made them perfect targets for our aim of trying to push the regressing mammary epithelial cells to die.
In addition to the inhibition of these molecules, we decided to directly interfere with the
polarization of the epithelial cells preventing the formation of adherence junctions. These
junctions as shown in the figure, Fig, are created by the Cadherins, a class of type-1
transmembrane proteins that play important roles in cell adhesion, forming adherence junctions to
bind cells within tissues together. Blocking the E-cadherins (E-cad) with an inhibitory antibody, they are not able anymore to form adherence junctions, and the cells lose an important structural anchor.
3.11 Test in vivo for the treated cells: Rag1-/- mice and transplantation
As mentioned before, we decided for explained reasons, to culture mammary gland cells in vitro.
These cells have been grown, induced to form tumors, and treated with different inhibitors, in order to try to reduce their predisposition to relapse. Thus, to investigate the potentialities of these cells to eventually regrow, giving rise to normal or tumorigenic mammary glands, it is fundamental to go back to the in vivo approach.
Indeed, the microenvironment is essential for the mammary gland development, due to the numerous signals received from the fibrous and adipose surrounding stromal cells.
The mouse mammary fat pad that hosts the mammary gland trees serves also as an orthotropic site for breast cancer.
The host microenvironment, in fact, influences breast development as well as tumor biology, affecting parameters such as gene expression, angiogenesis, growth, invasion, metastasis, and responses to therapy.
Consequently, having tumor models growing in appropriate orthotopic locations is necessary to obtain a rigorous understanding of tumor pathophysiology and to correctly study antitumoral treatments.
What is known is that both normal and prenoplastic mammary epithelia are stroma dependent and they only grow in mammary fat pad and not in ectopic sites.
Mouse mammary tumors are obviously not mammary stroma dependent because tumors can grow after subcutaneous implantation of tumorigenic cells. However we do not know if the ectopic tumor growth caused changes in the nature of the tumor cells. In addition, if the tumorigenic cells are transplanted directly into the fat pad, the tumor requires ten fold fewer cells to grow.
Tumor cells are stimulated by the presence of normal mammary epithelium in the fat pat.
In contrast, normal mammary epithelial cells are inhibited in their growth by the presence of pre- existing mammary epithelium in the fat pad. For this reason prior to any cell transplantation in the fat pad, it must be cleared from glandular elements. Another reason is the necessity to distinguish between transplanted and original cells in order to follow the eventual mammary gland growth derived from the transplanted ones.
Consequently, at 21 days old (Fig.3.17), when the branches of the mammary gland are not even reaching the lymph node, the portion of fat pad containing the branching is been cut off, leaving just the fat pad necessary for the further injection (Fig3.18).
Fig.3.17 Mouse mammary gland branching in the fat pad.
Fig.3.18 Clearing process of 21 days old mouse. The fat tissue containing the mammary gland branches is cut off until the lymph node, in order to only leave fatty tissue as support for further cells transplantations.
