Transformation of Normal HMECs (Human Mammary Epithelial Cells) into Metastatic Breast Cancer Cells: Introduction - The Broad Picture:

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1 Transformation of Normal HMECs (Human Mammary Epithelial Cells) into Metastatic Breast Cancer Cells: Introduction - The Broad Picture: Spandana Baruah December, 2016 Cancer is defined as: «A disease caused by an uncontrolled division of abnormal cells in a part of the body». Under normal circumstances, human mammary epithelial cells (HMECs) have a finite life span, they are not resistant to cell death and they do not undergo extemporaneous immortalization without any external stimuli. Normal HMECs undergo replication through cell division, these cells then undergo differentiation which then leads to the formation of various organs and organ systems (in this case, it leads to the formation of the mammary organs). In cancer cells however, this process of cell division does not occur the way it usually does in other HMECs. Here, the proteins and enzymes that are responsible for the sequential regulation of the process of cell division no longer properly drive the progression from one stage of the cell cycle to the next due to damaged DNA caused by an array of DNA damaging mutagens. source: DNA damage can be caused due to both endogenous factors (such as endogenous mutagens, spontaneous DNA changes, replication errors and dietary factors) and exogenous factors (such as genotoxic chemicals, physical carcinogens and viruses). Once the DNA has been damaged,

2 a number of mutations occur, in the cellular genome which causes either a Gain-Of-Function (seen in mutated oncogenes) or a Loss-Of-Function (seen in mutated tumor suppressors). At this stage, it is important to ask what causes the uncontrollably high rate of cell division in cancerous HMECs. On a molecular level, the principal cause of this dramatic rise in the rate of cell division can be attributed to mutations in growth controlling agents (primarily, growth controlling oncogenes and tumor suppressors). In addition to this, there are also epigenetic changes (changes in gene expression of cells with a normal DNA sequence) due to DNA methylation, chromatin remodeling and microrna. The damage that is caused in the cellular DNA is due to cells being subjects to tumor initiators, followed by tumor promoters or just carcinogens (which contain both tumor initiators and promoters in it). While the tumor initiator damages the DNA, the tumor promoter takes charge of pushing the damaged cells into wild proliferation. The following image illustrates the sequential flow of events showcasing the role played by tumor initiators and promoters: source:

3 This results in cancer cells that divide at a rate that is far greater than normal HMECs thereby forming cancerous tumors that are insensitive to growth inhibition and are independent of growth promoting signals. Thus, cancer cells that go through the process of cell cycle unchecked, over time, form malignant tumors that divide uncontrollably; furthermore, these cancer cells also go on to spread cancerous tumor cells throughout the rest of the body by the process of metastasis. Hence, putting all of these processes together, in order for a normal human mammary epithelial cell to completely transform itself into a metastatic cancer cell, it needs to fulfill the six essential characteristics or hallmarks of a cancer cell, namely: self sufficiency in growth signals, insensitivity to growth inhibition, immortalization (in terms of replicative power), resistance to cell death, angiogenesis (development of new blood vessels) and tissue invasion and metastasis. Thus, the journey of transformation of a typical HMEC into a human breast cancer cell will involve the sequential development of all of the above listed hallmarks, thereby turning itself into a fully metastatic, independent cancel cell. How is self sufficiency in growth signals attained by cancerous HMECs? Normal cells generally require growth factors to be externally produced; however, in the case of cancerous tumor HMECs, the cell either takes up the responsibility of producing its own growth factors or it becomes hyper-sensitive to its surrounding growth-factor levels. The other mechanisms through which sustained proliferation can be achieved by cancerous HMECs include deregulating mitotic signals, constitutively activating mitotic signaling pathways and disrupting negative feedback mechanisms ( cancer/growth_signals.html). Thus, simply put, the activation of mutations or the over expression of any component in the following pathway can lead to growth independence in cancerous HMECs: (Growth Factor (EGF) -> Growth Factor Receptor (EGF-R) > Transducer (RAS) > Effector (RAF) > Proliferation Factors (CDKs/cyclins). In the specific case of breast cancer, it is fairly common to see the activation of the H-RAS oncogene in order to achieve self sufficiency in growth signals. How do cancerous HMECs become insensitive to growth inhibition? In normal HMECs, powerful cell division controlling negative regulators control the process of cell proliferation. In cancerous HMEC s, in spite of the presence of strong anti growth signals by negative regulators, uncontrolled cancer cell proliferation is not terminated. This can be due to the inactivation of tumor suppressors such as Rb, p53, or TP53 or because of the evasion of contact inhibition mechanisms. In normal HMECs we see the production of CDK (cyclin dependent kinase) cell cycle inhibitors due to TGF. (transforming growth factor-beta). However, in cancer HMECs, a transducer protein called SMAD4 is mutated due to the decreased expression of TGF.-R (transforming growth factor-beta receptor) resulting in dedifferentiation and invasiveness, thereby rendering the HMEC insensitive to growth inhibition. How do cancerous HMECs render themselves as immortal in terms of replicative power? In order to overcome the senescence checkpoint that usually define the replicative life span of normal HMECs, oncogenic SMECs need to render themselves as immortal in terms of replicative power. This is can be achieved by a number of methods, some of them being:

4 chemical exposure, radiation exposure, over expression of certain viral oncogenes. In the specific case of the immortalization of oncogenic HMECs, the most effective way to achieve virtually unlimited replicative power is by the expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Additionally, the complete oncogenic transformation of normal HMECs requires multiple gene product expression, such as SV40 large T and small t, htert (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). Additionally, chromosomal telomeres play a very important role in the immortalization of cancerous HMECs. Telomeres are specialized DNA protein structures at chromosomal ends that are essential for cellular reproduction. Under normal circumstances, during each DNA replication round, around 200 nucleotides of telomeric DNA is lost - thus, in normal HMECs, the replication limit is rounds due to critical telomerase shortening. However, due to the process of lagging DNA strand synthesis, (specifically its reverse transcriptase function) telomerase is restored, thereby enabling oncogenic HMECs to act as immortal. source:

5 How do oncogenic HMECs attain resistance to cell death? As opposed to normal cells, cancerous HMECs are under constant stress, such as oncogenic stress, genomic instability, and cellular hypoxia. These act as internal stimuli triggering the process of apoptosis which is a cellular suicide program that is meant to eliminate unnecessary and unhealthy cells from the body. However, in order to continue replicating and thus forming breast cancer tumors, cancerous HMECs actively try to evade the process of apoptosis. The cancerous HMECs disable apoptotic pathways by employing strategies such as altering the expression of pro- and anti-apoptotic regulators or survival signals or by the loss of the TP53 tumor suppressor. It has also been experimentally shown that the inactivation of a BH3-only protein or a caspase not only aids cancerous HMECs in evading pro-apoptotic pathways but also accelerated the formation of tumors. How do cancerous HMECs promote the process of angiogenesis in its tumors? Angiogenesis is defined as the process of formation of new blood vessels; this process is naturally for maintaining cell growth and cellular healing in normal HMECs. However, in cancerous HMECs, the progression process of angiogenesis is used to recruit new blood source:

6 vessels in order to supply oxygen and nutrients that are required for the survival of the breast cancer tumors. Angiogenesis can be activated by altering the balance of inducers and inhibitors that normally keep this process tightly regulated. Principally through the signaling of VEGF (vascular epithelial growth factor), cancer tumors promote the process of angiogenesis. How do cancerous HMECs promote the process of metastasis? The process of metastasis is defined as: «Metastasis is the spread of cancer cells to new areas of the body (often by way of the lymph system or bloodstream). A metastatic cancer, or metastatic tumor, is one which has spread from the primary site of origin (where it started) into different area(s) of the body.» This process of metastasis is observed in attest 20-30% of breast cancer cases and it can occur up to 20 years before the diagnosis of breast cancer. Although the cancerous HMECs spread across the body to various organs, the cancer will still be classified as breast cancer due to the primary site of metastasis. Conclusion - What to expect once a normal HMEC has been transformed into a metastatic human breast cancer cell: Thus, by sequentially achieving all the six major essential hallmarks of cancer, normal HMECs are transformed into cancerous HMECs that promote the growth of cancerous tumors. After these HMECs have transformed completely, they begin to show signs of lacking differentiation, their structure appears to be atypical, their growth is erratic, they exhibit properties of invasiveness and they start to infiltrate their surrounding tissue. Once these properties are observed in a human mammary epithelial cell, it can be confirmed that the process of transformation of the HMEC into a metastatic breast cancer cell is complete.

7 References: 1. All lecture handouts BIOL Brown University