Lynch’s No1: Hypothesis: Ferroptosis can be triggered in Metastatic Uveal Melanoma patients by increasing cellular IRON and using the ferroptosis inducing drug ERASTIN.

Abstract: Uveal or Ocular melanoma (OM) has a very poor prognosis when it spreads. There is no known cure. A correlation between low iron has been noted with metastatic progression. Unusual OM remission cases have been connected with ferroptopic inducers. Zinc has also been connected with OM and ferroptosis. The drug erastin is known to induce ferroptosis. Ferroptosis is iron dependent. BAP1 loss in OM is known to inhibit ferroptosis. The combination of these factors presents a novel opportunity to treat this disease with erastin and increased iron.

Background: Uveal or Ocular melanoma (OM) is a rare cancer of the eye. Incidence rates are circa 5.1 per million (USA) to as high as 9.5 per million (Ireland) [1] . It can be treated with some success if detected early by radiation or removal. However when it metastasises (50% rate) it has a record of being lethal with death in 2-9 months, a figure holding steady these past years. It has proven not to be responsive to traditional standards of treatment [2].

It has become very popular in this internet age for patients and those around them to search the published online medical literature for items of OM interest. Recently a paper on the therapeutic use of Zinc in cancer was unearthed [3]. More interestingly to this author is that a successful case reported was that of a uveal melanoma patient. This raised the question why did Zinc (Zinc gluconate (50 mg chelated elemental Zn2+)) work in such a fashion.

Zinc intoxication of cells induces ferroptosis in non-small cell lung cancer (NSCLC) cancer cells [4]. Zinc’s relationship with H2O2 is of interest here and raises questions of similarities with the action of acerbic acid (Vitamin C) injections.

Ferroptosis is a new type of cell death that has been discovered in recent years and is usually accompanied by a large amount of iron accumulation and lipid peroxidation during the cell death process; the occurrence of ferroptosis is iron-dependent. Ferroptosis inducing factors can directly or indirectly affect glutathione peroxidase through different pathways, resulting in a decrease in antioxidant capacity and accumulation of lipid reactive oxygen species (ROS) in cells, ultimately leading to oxidative cell death.

On a parallel search evidence was discovered about Artesunate (ART) which is a derivative of artemisinin, the active principle of the Chinese herb Artemisia annua L. Artesunate is approved for the treatment of multidrug-resistant malaria and has an excellent safety profile. It has been shown that Artesunate, apart from its anti-malarial activity, has cytotoxic effects on a number of human cancer cell lines. This report featured an OM patient still alive after 47 months with a median survival of 2-5 months while being treated with ART [5]. ART is described as a ferroptopic agent.

By way of anecdotal evidence from the OM community it has been observed that those who die of metastatic OM tend to have very low iron levels. Those that are living longer tend to have normal iron levels. As ferroptosis requires iron, supplementation for anaemic patients would seem to be necessary. Consideration should be give to iron infusions and or blood transfusions to help achieve the ferroptopic iron levels required.

Loss of the tumour suppressor BRCA1-associated protein 1 (BAP1) is an indicator of metastases in OM [6]. BAP1 inhibits cystine uptake by repressing SLC7A11 expression, leading to elevated lipid peroxidation and ferroptosis [7].

System xc− was recently described as the most upstream node in a novel form of regulated necrotic cell death, called ferroptosis. In this context, the small molecule erastin was reported to target and inhibit system xc−, leading to cysteine starvation, glutathione depletion and consequently ferroptotic cell death. Although the inhibitory effect of erastin towards system xc− is well-documented, nothing is known about its mechanism of action. System xc− is one among many amino acid transporters expressed in the plasma membrane of mammalian cells. This transporter is composed of xCT (SLC7A11), which is the substrate-specific subunit hence the BAP1 loss connection[8].

The drug Erastin triggers oxidative, iron-dependent cell death. Cell death triggered by erastin is significantly inhibited by antioxidants (e.g., α-tocopherol, butylated hydroxytoluene, and β-carotene) and iron chelators, suggesting that ROS- and iron-dependent signaling is required for erastin-induced ferroptosis. Erastin can directly bind to VDAC2/3 in BJeLR cells. Knockdown of VDAC2 and VDAC3, but not VDAC1, leads to erastin resistance. Erastin has the ability to reduce glutathione level by directly inhibiting cystine/glutamate antiporter system Xc− activity, with activation of the ER stress response. Erastin shows a dose-dependent effect, and 30 μM of erastin displays the most dramatic effect[9].

2020 has seen some exciting new research published. One such paper “The epigenetic regulators and metabolic changes in ferroptosis-associated cancer progression” [10] has surfaced. Its a very dense work brimming with information which inter alia connects OM and loss of BAP1 with ferroptosis disruption.

Hypothesis:

It is hypothesised that:

• Metastatic Uveal Melanoma cells can be killed by a process called ferroptosis.
• Available Iron at the cellular level is needed.
• For speed infusions and transfusions should be considered to meet this iron requirement.
• Zinc levels should also be investigated and acted on if appropriate.
• The ability of the body to trigger ferroptosis may be inhibited and may need help.
• The drug Erastin induces ferroptosis but needs iron to be available.
• Its use with increased iron levels should be evaluated firstly in a BAP1 negative metastatic uveal melanoma setting.

No evidence of erastin use in OM has been found in the literature in this exercise. Consideration should be giving to known erastin inhibitors ( LSH, NFS1, GPX4 , SCD1 and FADS2) and their impacts with the suggested patient profile. In the absence of any known viable working therapy this hypotheses should be tested at the earliest opportunity. In particular where all options have been exhausted and the “Right to try before I die” argument comes into play, it should be considered.

Declaration of Competing Interest:

The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author:

Mr. Brendan Lynch is the surviving spouse and carer of a deceased uveal melanoma patient Ms. Belinda Honeyman-Lynch. He has continued the passionate search for a cure. He is an active member in the OM community at national and international levels. He is also a founding member of Ocular Melanoma Ireland (www.ocumirl.org). He is a retired civil servant who's only declared interest is the discovery of a cure for ocular melanoma.

Bibliography

1: Caroline Baily, Valerie O’Neill, Mary Dunne, Moya Cunningham, Giuseppe Gullo, Susan Kennedy, Paul M. Walsh, Sandra Deady, Noel Horgan, Uveal Melanoma in Ireland https://www.researchfoundation.ie/app/uploads/2019/05/Uveal-Melanoma-in-...,

2: Jessica Yang, Daniel K. Manson, Brian P. Marr, and Richard D. Carvajal, Treatment of uveal melanoma: where are we now? https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824910/,

3: , Unlocking Zinc’s Potential to Fight Cancer. https://www.cancertreatmentsresearch.com/unlocking-zincs-potential-to-fi...,

4: , Zinc intoxication induces ferroptosis in A549 human lung cells. https://www.ncbi.nlm.nih.gov/pubmed/30968088,

5: Berger TG1, Dieckmann D, Efferth T, Schultz ES, Funk JO, Baur A, Schuler G., Artesunate in the treatment of metastatic uveal melanoma--first experiences. https://www.ncbi.nlm.nih.gov/pubmed/16273263, 2005

6: , Clinical Characteristics of Uveal Melanoma in Patients With Germline BAP1 Mutations. https://www.ncbi.nlm.nih.gov/pubmed/25974357,

7: , BAP1 links metabolic regulation of ferroptosis to tumour suppression. https://www.ncbi.nlm.nih.gov/pubmed/30202049,

8: , The ferroptosis inducer erastin irreversibly inhibits system xc− and synergizes with cisplatin to increase cisplatin’s cytotoxicity in cancer cells. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772355/,

9: , Erastin is a ferroptosis inducer. Erastin binds and inhibits voltage-dependent anion channels (VDAC2/VDAC3). https://www.medchemexpress.com/Erastin.html,

10: , The epigenetic regulators and metabolicchanges in ferroptosis-associated cancerprogression. https://molecular-cancer.biomedcentral.com/track/pdf/10.1186/s12943-020-... ,

Lynch’s No2 Hypothesis:  Iron is needed for Ferroptosis but its availability can be inhibited at many points. A broad scoping & attack approach is warranted to make iron available for ferroptopic action.

Abstract:  Cancers like Uveal / Ocular melanoma (OM) have a very poor prognosis when they spread. There is no known cure. A correlation between low iron has been noted with metastatic progression. Unusual OM remission cases have been connected with ferroptosis & ferroptopic inducers. Iron is needed for ferroptosis, here we explore the low availability of iron at the cellular level, what  the causes may be and possible therapeutic options.

Background: It is well know that iron deficiency or over exposure has noticeable effects on human health. Iron is an essential element for almost all living organisms as it participates in a wide variety of metabolic processes, including oxygen transport, deoxyribonucleic acid (DNA) synthesis, and electron transport [1].  Iron is delivered to tissues by circulating transferrin, a transporter that captures iron released into the plasma mainly from intestinal enterocytes or reticuloendothelial macrophages[2]. Enterocytes can be killed by rotavirus and suffer other injuries. Iron absorption into the plasma is controlled by ferroportin. 

Hepcidin is a circulating peptide hormone secreted by the liver that plays a central role in the regulation of iron homeostasis. It is the master regulator of systemic iron homeostasis, coordinating the use and storage of iron with iron acquisition [3].

Ferritin stores iron, representing iron status. Hepcidin binds to ferroportin, thereby inhibiting iron absorption/efflux. Inflammation in CKD increases ferritin and hepcidin independent of iron status, which reduce iron availability[4]. ccRCC is a sister clear cell cancer to OM and others. These cancers are susceptible to ferroptosis. In the absence of anything in the literature it is hypothesised that hepcidin acts the same way in these cancers and possibly more types.

Dysregulation of hepcidin can lead to iron disorders. In this paper we are primarily concerned with iron not being available at the cellular level for ferroptosis as needed in cancer cell control. It is hypothesised that hepcidin plays a key role and one that has not yet been tested on OM patients.

Increased hepcidin is associated with  chronic inflammation, antimicrobial activity (Escherichia coli (ML35P), Neisseria cinerea, Staphylococcus epidermidis, Staphylococcus aureus & Streptococcus agalactiae) and with action against the fungus Candida albicans. Interestingly hepcidin benefits from hypoxia.

Anecdotally OM patients have been observed with anaemia and others with high ferritin levels which is confusing. However if we consider the role of hepcidin we may hypothesise that both cohorts have reduced iron available for ferroptosis.

Inhibitors of hepcidin are erythroferrone [5] (no draggable candidate found in this search) and large doses of vitamin D (100,000 IU vitamin D2)  [6].

Iron excess can lead to the generation of reactive oxygen species (ROS) which can cause cellular oxidative stress and induce apoptosis in cancer cells. Lack of or lowered iron levels can inhibit  ROS an thus allow cancer cells to progress unchecked.

Iron is highly conserved by the body [7] with the main losses in women with excessive menstrual blood loss.  Major inhibitors of iron absorption are phytic acid, polyphenols, calcium, and peptides from partially digested proteins. Enhancers are ascorbic acid and muscle tissue which may reduce ferric iron to ferrous iron and bind it in soluble complexes which are available for absorption [8]. This an interesting point as ascorbic acid (vitamin C) is regularly injected into cancer patients for its ROS generating properties.
On the inhibitor side black tea has been identified as a polyphenol. Ireland is the second highest consumer of black tea in the world behind Turkey.

Another point of note is the competition with iron for absorption by other heavy metals. Of major interest here is lead. Lead is taken up by the iron absorption machinery (DTM1), and secondarily blocks iron through competitive inhibition. Ireland has areas where public tap water is up to 15 x the national safety limit. The WHO has stated that there are NO safe limits for lead in drinking water[9]. 

Iron deficiency can exist with or without anemia. Some functional changes may occur in the absence of anaemia, but the most functional deficits appear to occur with the development of anaemia. Even mild and moderate forms of iron deficiency anaemia can be associated with functional impairments affecting immunity mechanisms [10],

Testing for available iron levels in serum can be problematic. The WHO has recommended that haemoglobin and ferritin, a protein that is strongly correlated with iron stores in healthy people, are the most useful indicators of the impact of programmes to control iron deficiency. The main problem with ferritin lies in the fact that it is affected by inflammation due to infection and chronic disease, so it is less useful to assess the prevalence of iron deficiency than to estimate a change brought about by a programme. Because of this, the WHO recommended that to assess iron deficiency, the transferrin receptor, in addition to haemoglobin and ferritin, should be measured in places where infection is common[11].

Zinc protoporphyrin reflects the shortage of iron supply and can be detected by RBC fluorimetry.

Anaemia is where the number of RBCs in the blood are low. It is not a measure of iron but is more an indicator of low iron and disease process. Iron is the leading cause of anaemia but not the sole cause. Other causes can be chronic disease, active bleeding, kidney disease, pregnancy, low vitamin B12, A, folate ribiflavin and copper.  

Obesity may be connected to low haemoglobin concentration low serum iron, transferrin saturation band elevated serum ferritin. Obesity-related inflammation may increase hepcidin concentrations and reduce iron availability [12]

Other causes of anaemia are alcoholism, sickle cell, thalassemia, aplastic & hemoltic

Hypothesis:
It is hypothesised that:

    • Iron is necessary for ferroptosis in OM. 
    • That iron must be available at the cellular level.
    • Available Iron levels should be measured appropriately (WHO guidelines).
    • A supportive iron environment should be established.
    • Immediate response should go direct to parenteral iron treatment. 
    • Hepcidin a master iron controller should be managed aggressively.
    • Lead levels in the patient should be tested and addressed
    • In restoring the bodies natural iron pathway post primary treatments, causes of damage must be fully addressed. 

Failure to cover all the escapes & blocks will result in failure. To succeed treating physicians will have to be thorough. 

Declaration of Competing Interest:
The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The author:
Mr. Brendan Lynch is the surviving spouse and carer of a deceased uveal melanoma patient Ms. Belinda Honeyman-Lynch. He has continued the passionate search for a cure. He is an active member in the OM community at national and international levels. He is also a founding member of Ocular Melanoma Ireland (www.ocumirl.org). He is a retired civil servant who's only declared interest is the discovery of a cure for ocular melanoma. 

Bibliography
1: Nazanin Abbaspour, Richard Hurrell,1 and Roya Kelishadi2, Review on iron and its importance for human health, , https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999603/#!po=18.7075
2: David M. Frazer, and Gregory J. Anderson, Iron Imports. I. Intestinal iron absorption and its regulation, , https://journals.physiology.org/doi/full/10.1152/ajpgi.00220.2005
3: Elizabeta Nemeth 1, Tomas Ganz, Regulation of Iron Metabolism by Hepcidin, , https://pubmed.ncbi.nlm.nih.gov/16848710/
4: Norishi Ueda1,* and Kazuya Takasawa2,3, Impact of Inflammation on Ferritin, Hepcidin and the Management of Iron Deficiency Anemia in Chronic Kidney Disease, , https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6163440/
5: Mark J. Koury, MD, Erythroferrone: A Missing Link in Iron Regulation, , https://pub.hematology.org/Thehematologist/Years-Best/3599.aspx
6: Justine Bacchetta,*†‡ Joshua J. Zaritsky,† Jessica L. Sea,* Rene F. Chun,* Thomas S. Lisse,* Kathryn Zavala,* Anjali Nayak,† Katherine Wesseling-Perry,† Mark Westerman,§ Bruce W. Hollis,‖ Isidro B. Salusky,† and Martin Hewisoncorresponding author*, Suppression of Iron-Regulatory Hepcidin by Vitamin D, , https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3935584/
7: Janet R Hunt 1, Carol Ann Zito, LuAnn K Johnson, Body Iron Excretion by Healthy Men and Women, , https://pubmed.ncbi.nlm.nih.gov/19386738/
8: Richard Hurrell 1, Ines Egli, Iron Bioavailability and Dietary Reference Values, , https://pubmed.ncbi.nlm.nih.gov/20200263/
9: Dr G Stewart, Special Report by Dr Stewart2019 - Lead in Ireland, , https://www.drstewartsclinic.com/2019-lead-in-ireland
10: Mark L. Failla, Trace Elements and Host Defense: Recent Advances and Continuing Challenges, , https://academic.oup.com/jn/article/133/5/1443S/4558523
11: , WHO/CDC expert consultation agrees on best indicators to assess iron deficiency, a major cause of anaemia, , https://www.who.int/mediacentre/news/notes/2004/anaemia/en/
12: I Aeberli 1, R F Hurrell, M B Zimmermann, Overweight Children Have Higher Circulating Hepcidin Concentrations and Lower Iron Status but Have Dietary Iron Intakes and Bioavailability Comparable With Normal Weight Children, , https://pubmed.ncbi.nlm.nih.gov/19636315/