The Role of Telomeres in Cancer Development and Progression, and the Double Edge Sword Effect with Tamoxifen

Th e current practice regarding the use of estrogen receptor antagonists in breast cancer, (such as Tamoxifen), has opened doors for investigators to research these drugs for their use in the prevention of breast cancer. Although this approach is suggested by the literature to be an eff ective strategy, currently there are no established guidelines applicable to the use of anti-estrogen receptor drugs or prevention in the clinical setting [1,2].


Introduction
Th e current practice regarding the use of estrogen receptor antagonists in breast cancer, (such as Tamoxifen), has opened doors for investigators to research these drugs for their use in the prevention of breast cancer. Although this approach is suggested by the literature to be an eff ective strategy, currently there are no established guidelines applicable to the use of anti-estrogen receptor drugs or prevention in the clinical setting [1,2].
We review the literature related to Tamoxifen therapy and recommend expansion of the application of low dose Tamoxifen for patients with positive estrogen receptor cancers to preventive oncology. Th ese applications include patients at risk for hormonally responsive cancers, and expand beyond that to individuals who may not be even at risk as defi ned by current risk stratifi cation standards, such as strong family history of breast cancer. In this article we also review an opposing negative eff ect of Tamoxifen in patients with active cancer, and discuss why we believe it should be "avoided" in the treatment of solid tumors (except breast cancer), although it may be a meaningful tool in prevention of cancer of all types.

Background
Th e double edge sword of Tamoxifen (favorable in prevention and unfavorable in therapy), is based on our novel understanding of telomeres and their role in carcinogenesis and cancer progression [3][4][5][6]. Further studies are warranted in application of low dose Tamoxifen to prevent all solid tumors in unaff ected individuals, in a safe and effi cacious manner. Th e theory of how Tamoxifen interacts with telomeres also provides a basic understanding of possible unfavorable mechanisms involved with Tamoxifen's application and its resistance in treated tumors.

Telomeres and Tamoxifen
Tamoxifen is shown in the literature to be a telomerase activator [7]. Th e activation of telomeres is an important step in carcinogenesis as it is secondary to what is known as a "telomere crisis" [3,8]. As we age, the length of the telomeres shortens. Continuation of this process over time is important, as it causes the cell DNA to be exposed to genomic instability. Th is instability at a critical length of telomeres promotes apoptosis, by activating the P53 and other onco-suppressor genes in normal circumstances.
However, if this instability continues beyond a certain point, and/ or if the P53 system is impaired, or if the cell is exposed to a telomerase activator (such as Tamoxifen), it can cause DNA damage and mutations in both oncogene promotors as well as oncogene suppressors, which can promote the cancer development at the fi rst stages (carcinogenesis). It is therefore postulated that the telomere crisis happens long before the DNA mutations required for carcinogenesis. Th e unstable DNA shares the same alterations identifi ed in cancer. Finally, activation of telomerase can save the cell from apoptosis, and death. Th is activation is uniform in almost all cancer cells, with high proliferative index [4] shown by both tumor tissue biopsy as well as liquid biopsies. Although it is suggested that telomerase activity is not required for malignant transformation, and it merely an index for highly proliferative cells, it is commonly used as a marker for detection and monitoring the cancer cells, both in tissue biopsies as well as circulating tumor cells or CTCs (Telomerase is an indicator for presence of tumor epithelial cells in the blood) [9,10].
Studies have shown that by using tamoxifen in a cellular culture, the ability of the cells to activate telomerase increases, and as the result the cell will continue to stay young, with prolonged resistance to crisis [7]. Th is can prevent the cell from an increased amount of genomic instability and further carcinogenesis. However, the same mechanism can explain the cancerous cell's ability to survive longer in the presence of Tamoxifen, as a telomerase activator [7]. Th e cancer cells exposed to 4-hydroxy Tamoxifen in culture are able to survive longer and infi ltrate distant organs in mice studies, explaining the increased invasion. Th e critical question remains whether the application of Tamoxifen as early as possible in individuals not known to have cancer may prevent the telomere crisis, and if so, it can prevent carcinogenesis long before it is clinically diagnosed.
Tamoxifen therapy also may have unintended eff ects on gene expression. Th is theory is presented in the following two cases studies, both patients with breast cancer who had positive circulating tumor DNA for Neurofi bromatosis-I gene post tamoxifen therapy.

Case Studies
Breast cancer with positive NF-1 70 year old female with history of left breast ductal carcinoma diagnosed in 2001 status post lumpectomy and hormonal therapy. Hormonal therapy consisted of Femara for 3 years and Arimidex for 4 years. She was also treated with radiation, and was status-post recurrence of the tumor in same side documented in September 2013, through core biopsy and referral for surgery (mastectomy), and chemotherapies. Th e extent of disease was documented to involve the skin and required neoadjuvant chemotherapies. Her left breast had a 5 cm mass. She was seen and evaluated for complimentary medicine, and immediately started on IV epigenetic therapies aft er initial labs. Her initial labs showed extensively elevated angiogenic markers, including Interleukin 8 (both in serum and plasma). Her Interleukin 8 was 103.3 in plasma (normal less than 34) and 136 in serum (normal less than 66), measured on 6/3/14. Treatment was given every day in fi rst two weeks, fi ve days a week, and twice a week aft er. She was started on Tamoxifen at a dose of 5 mg daily. Her labs were repeated on 6/17/14 and 8/29/14. She reported improved QOL while receiving therapies and no side eff ects experienced. Her labs showed marked reduction in both markers. Her IL-8 in serum dropped down to 89 and in her plasma down to 16.8. Further this dropped down to normal range. Her Interleukin 8 normalized both in serum as well as plasma (50 in serum and 27 in plasma), on 10/8/2014. She subsequently underwent surgery on 12/14, and currently is in remission. She received Firmagon injection, as well as Cytoxan (metronomic dose) along with our therapy, and her VEGF dropped down to less than 9, on 5/28/15. Her circulatory tumor DNA (ctDNA) was studied through Guardant 360 Lab, on 6/25/15, which showed positive NF-1, and APC, likely due to the exposure to anti-estrogen therapy. Following further treatments with MTET (Multi-targeted Epigenetic therapy), her Guardant 360 was repeated and showed no ctDNA (Figure 1).
Th is anti-angiogenetic response to the therapies in a novel approach needs to be investigated further in clinical trials to reduce the risk of recurrence and improve response to conventional treatments. and axial lymph node resection in November 2012, which was positive for 2 LN invasions. Upon her arrival at our clinic, she had recurrence of her tumor, with two large palpable sites in the chest on skin. She was complaining of a new onset consistent and dry cough, and was concerning for lung metastasis. Upon evaluation, we ordered a PET scan, which showed multiple pulmonary metastasis bilaterally, which were too numerous to count, with an SUV max of 4.8. She had multiple masses in right breast with max SUV of 12.9, and multiple smaller lesions with max SUV of 5.5, plus mediastinal and internal mammary Breast cancer with positive NF-1 43 year old female with history of breast ductal carcinoma, 100 percent ER and PR positive, and a Her-2 negative. Her Ki67 was 80 percent; SBR on biopsy was determined to be 8/9. She had exhausted many alternative cancer therapies since 5/2012, when she was diagnosed fi rst. None of the therapies was eff ective, and in fact her cancer grew to 4 cm, and she opted for bilateral mastectomy The chart above annotates the percentage or allele frequency , of altered circulating cell free-DNA (% cfDNA ) detected in this patient. The detected genomic alterations arelisted in descending order by % by gene.
The "FDA Approved in indication "and " Available for use in other indications" columns describe drugs associated with specific fenomic alterations. It based on publicly available information as described in the " Detailed Therapy Results" and "clincial Relevance of Detected Alterations" sections of the report incidence of endometrial and ovarian cancer [11,12]. We also recommend large scale studies using Tamoxifen for prevention of cancer. Perhaps the most important question that remains to be answered is the timing of Tamoxifen administration in the prevention of cancer. LNs in chest and supraclavicular LNs. In additional she had multiple metastatic LNs in right axilla with SUV max of 2.9.
She had tumor markers all elevated in labs. Immediately she was started on IV epigenetic therapies which she received on daily basis. Aft er 10 treatments, her labs were rechecked. Th e results showed signifi cant decreases in all her tumor markers. Her cough improved, and she maintained her treatments at our clinic. She did not change her diet, nor did she start any chemotherapy. At this time, she was not on any hormonal blockade, confi rming the independent response to epigenetic therapy. In November 2014, she had a restaging PET scan, showing marked reduction of the activities in mediastinal and internal mammary LNs, with improved diff use pulmonary metastasis. Th e PET scan was performed and compared with PRE treatment scan aft er 6 weeks of therapy. Th e following PET scan done on 1/12/15, demonstrated complete resolution of all pulmonary metastasis and axillary/supraclavicular lymph nodes. Th ere was minimal residual disease reported in the mediastinal lymph nodes, with SUV activity decreased to 5 from 10.6, and to 3 from 7. At this time, the tumor markers were substantially reduced and most returned to normal limits. CA 15. 3  . She was restaged again with a PET/CT on 4/15/2015, which showed progression of the local disease in surgical incision, but only minimal residual disease and faint FDG activity (SUV activity of 1.5 compared to 4.8 pre-treatment) in her mediastinal pulmonary lesions, with complete resolution of liver fi ndings. Due to the fact that she was down staged from a Stage IV disease to a local disease in the breast, it was justifi ed to remove the breast tissue and treat her locally with surgery.
Following her surgery, her circulatory tumor DNA was evaluated through Guardant 360, which revealed positive EGFR. Subsequently the patient was started on Tarceva, and the Guardant 360 test was repeated. Although the EGFR mutation allele fraction (MAF) decreased with therapy, the second test showed NF-1, ALK, EGFR and cMET ( Figure  2). Th e presence of NF-1 confi rmed the mutated oncosuppressor gene, likely due to the exposure to anti-estrogen therapy, an example of induced heterogeneity. Th e presence of other alterations, confi rmed the induction of heterogeneity by Tarceva. Th e presence of NF-1 and other alterations, although considered poor prognostic markers, did not change this patient's outcome, or her disease free survival. We believe that this success was due to the combination of epigenetic therapies.
Th is case suggests an independent, as well as synergistic method of epigenetic therapies combined with hormonal blockade, which was able to treat non-operable Stage IV breast cancer, and down stage the patient successfully to treat the local disease. Th is quick response suggests further need to evaluate this novel combinational approach in treating resistant and recurrent advanced breast cancer.

Conclusion
Tamoxifen has been proven to prevent invasive and noninvasive breast cancer [1,2]. Retrospective studies also indicate an eff ect on