In vitro cobalt-stimulated hypoxia-inducible factor-1 overexpression does not correlate with cancer risk from cobalt exposure in humans

Abstract

Chemical-induced pulmonary carcinogenesis in humans is usually a multi-decade process. The average age at diagnosis of lung cancer in cigarette smokers, and mesothelioma in asbestos workers, is approximately 70. The carcinogenic process consists of genetic changes to normal cells usually sequenced as initiation (mutations), promotion (clonal expansion of initiated cells), and progression (carcinoma in situ to invasive carcinoma to metastatic carcinoma). Angiogenesis, the sprouting of new vessels from preexisting capillaries, plays an important role in the progression of small avascular tumors to vascularized invasive carcinomas and metastatic carcinomas. While the overall carcinogenic process is multi-decade, the transition from avascular to vascularized carcinoma is believed to take place over the course of a much more limited time span. Tumor progression is a late-stage event in carcinogenesis. About 70% of human cancers including lung cancer express hypoxia-inducible factor (HIF)-1 in the absence of cobalt exposure. Cobalt compounds administered to transformed cell lines and primary cultures of human endothelial cells, smooth muscle cells, and mesenchymal stem cells can elicit overexpression of HIF-1. Cobalt-induced expression of HIF-1 would not be expected to interact with either the avascular initiation or promotion phases of carcinoma development. Humans exposed long-term to cobalt leaching from implants do not have an elevated cancer risk, and neither do goats ingesting up to 667 times the recommended daily human cobalt dietary requirement throughout their 15- to 18-year life span. In vitro hypoxia-mimetic characteristics of cobalt compounds do not correlate with the absence of increased risk following long-term systemic exposure to cobalt in humans.

Keywords

Cobalt hypoxia inducible factor cell culture angiogenesis late stage

Executive summary

Many thousands of patients with metal-on-metal hip implants that have begun to shed cobalt into the systemic circulation have not developed excess cancers over a time period of decades.

Despite regularly ingesting a cobalt level up to 667 times the recommended intake for humans, over their life span of 15–18 years goats display low cancer rates overall and do not develop tumors suggestive of exposure to a carcinogen.

National Toxicology Program (NTP) rodent 2-year bioassays suffer from high false-positive rates. Cobalt compounds administered via inhalation in an NTP 2-year rodent bioassay induce pulmonary tumors in rats. The well-established mechanism of reparative mitogenesis inducing mutagenesis described by Ames and Gold, Cohen and Ellwein, Moolgavkar, and Tomasetti and Vogelstein was not considered by the NTP as an underlying mechanism for the development of the rat tumors, but NTP instead posited that cobalt exposure was inducing hypoxia-inducible factor (HIF) thereby facilitating angiogenesis.

Rodent pulmonary tumors differ from human lung cancers in that the human tumors tend to be highly vascularized thereby possessing metastatic potential. In contrast, rodent pulmonary tumors tend to be localized and less vascularized. Inhalation of cobalt compounds in rats and mice does not model the late final progression stage of human pulmonary carcinogenesis but rather attempts to model the earlier stages of tumor initiation and promotion not affected by the overexpression of HIF.

The mouse two-stage dermal promotion assay provides a framework for conceptualizing the stages of cancer development as initiation (mutation), promotion (clonal expansion of initiated cells), and progression (angiogenesis, localized invasion, and metastasis).

Angiogenesis is a late change in tumor development usually occurring decades after the beginning of exposure to a carcinogenic agent or action. Tumors usually do not undergo angiogenesis until exceeding a diameter of 1–2 mm³.

Pulmonary inflammation produces a hypoxic local environment. High doses of inhaled cobalt compounds and many other pro-inflammatory agents induce pulmonary inflammation in rodents.

Over 70% of all human cancers are HIF positive in the absence of exposure to cobalt compounds.

In vitro, cobalt induces the expression of HIF in both transformed and normal cell lines. The majority of in vitro demonstrations of cobalt-induced HIF expression is reported in transformed cell lines. Transformed cell lines have already experienced one or more oncogenic mutations and are not analogous to normal cell lines.

The mechanism by which cobalt-induced overexpression of HIF-1 would be expected to influence cancer would be an increased tendency toward angiogenesis thereby accelerating the later progression stages of the disease. Evidence suggesting that this is actually occurring is absent. Therefore, the hypoxia-mimetic characteristics of cobalt compounds seen in in vitro cell culture studies do not correlate with the risk of exposure to cobalt compounds.

Very large numbers of patients with cobalt-shedding hip implants have not developed cancer

The epidemiological associations between occupational exposure to cobalt and cobalt compounds and risk of lung cancer are potentially confounded and unclear leading the National Toxicology Program (NTP) to consider the evidence from human studies to be “inadequate.”^1

–5 In industrial environments, the primary route of exposure to cobalt is via inhalation.^6

–9

Cobalt is rarely the only agent of potential concern found in workplace air in the facilities studied.^6,7 In addition, it is very difficult in these occupational cohorts to control for the potential contribution to risk from by far the most important pulmonary carcinogen, that is, cigarette smoking. Not only does smoking account for 80–90% of lung cancer cases,¹⁰ but the duration of smoking is more important than the intensity of smoking in terms of lung cancer risk.¹¹ Therefore, an accurate determination of both current and past smoking history would be essential toward disentangling a possible effect of smoking from cobalt or other inhalation exposures. A sufficient evaluation of current smoking status and smoking history is not available for the occupational cohorts exposed to cobalt compounds via inhalation.^6

–9

In contrast with occupational cohorts with exposures to cobalt confounded by other inhaled agents and indeterminate smoking histories, cobalt exposures from aging medical devices occur in the absence of co-exposure to agents other than chromium. Also, exposure to cobalt from aging medical devices has been occurring for decades.¹² More than one million metal-on-metal (MOM) prosthetic hips have been surgically implanted worldwide.¹² Several studies have demonstrated that as the surfaces of these devices wear a significant shedding into the general circulation of cobalt and chromium ions occurs.¹³ A number of studies on large numbers of patients have not found an increase in cancer incidence in association with these aging implants.^14

–20 Steinberg et al.¹³ studied epigenetic methylation changes in 34 osteoarthritis patients with MOM total hip replacements and 34 osteoarthritis patients with non-MOM total hip replacements and found no difference between these small groups. The results from this pilot study are consistent with the absence of cancer risk in MOM patients.

Very high lifetime (15–18 years) of ingestion of cobalt does not correlate with cancer rates in goats supporting the absence of tumors in MOM patients

Adjusted for body weight, goats have the highest mineral requirements of all ruminants. In proportion to body size, the goat’s metabolic rate is higher, and the surface area and capacity of the rumen is larger in comparison to the cow, sheep, or horse.²¹ Domestic goats are at greater risk for parasitical infestation than wild goats probably because the freedom of wild goats allows them to not browse the same area twice.²¹ Parasite burdens are associated with anemia in goats thereby increasing the need for cobalt to facilitate hemoglobin formation. Cobalt is directly involved in vitamin B12 formation by bacteria in the stomachs of ruminants. Sheep require more cobalt than cows. Goats have four times the cobalt requirement as do sheep. Goats have a dietary requirement for cobalt of 0.1–10 ppm per day.²² In all animals, cobalt displays relatively low toxicity. Cobalt toxicity in ruminants is rare because toxic levels are approximately 300 times dietary requirement levels. On average, ruminants have 10 times the amount of cobalt in their system as compared with dogs, cats, rats, or man. The recommended level in humans is 0.015 ppm.²³ The high end of the recommended level for goats (10 ppm) is 667 times the recommended level for humans.

Despite a relatively high intake of cobalt, goats do not show an increase in cancer incidence compared with other animals.²⁴ Across mammalian species, the risk of cancer depends on both the number of cells in the body and number of years over which those cells can accumulate mutations. Cancer incidence can be calculated as a function of Mass × Life Span. The goat has a log (Mass × Life Span) = 5.4. When necropsied for tumor incidence, about 1% of goat necropsies present with a tumor. According to Abegglen et al.,²⁴ this places the goat relatively close to the raccoon as regards the tendency toward cancer formation.

Oregon State University has studied the distribution of tumor sites in the 1% of goats that do develop tumors. At their Veterinary Diagnostic Laboratory, 1146 goat (caprine) necropsy or biopsy specimens were submitted from 1987 to 2011. Of the 1146 specimens, 100 goats (8.7%) presented with 102 tumors.²⁵ Histopathology was performed on all but three of the tumors, that is, 97 tumors. The most common tumor was lymphoma (n = 17) with most of the lymphomas being multicentric.²⁶ There were 10 cases of cutaneous squamous cell carcinoma and 9 cases of thymoma. Seven mammary neoplasms were all classified as adenocarcinomas. Seven vascular proliferations were reported with five being hemangiosarcomas. Four malignant melanomas were seen. These six tumor types constituted 55.7% of the total cancer cases in goats.

A number of rare tumors were also found among the 97 tumors in goats including the following: one choroid plexus carcinoma, two rhabdomyosarcomas, three pheochromocytomas, two histiocytomas, five mast cell tumors, one amyloid-producing odontogenic tumor, one myxosarcoma, one sebaceous carcinoma, one apocrine sweat gland adenoma, and one thyroid carcinoma. The results from this 25-year retrospective study conducted by Oregon State University demonstrate that while lymphoma and other tumors do occur in goats, the rate of tumor formation is low, and when a goat does develop a tumor, it tends to be only a single tumor.²⁵ The causation of lymphoma in goats is believed to have a genetic basis with a possible contribution from eating pesticides.²⁷ The squamous cell carcinomas in goats occur in body parts not well protected from sunlight and are consistent with the large amount of sun exposure goats receive over their life span of 15–18 years.^28,29 Despite regularly ingesting a cobalt level up to 667 times the recommended intake for humans, goats display low cancer rates overall and do not develop tumors suggestive of exposure to a carcinogen via the oral route.³⁰

NTP rodent 2-year bioassays suffer from high false-positive rates

Our group conducted a mechanistic and statistical analysis of the entire NTP rodent 2-year bioassay database up through 2017.^30

–36 In the 1980s, Cohen, Ellwein, and their colleagues conducted a series of studies that demonstrated that cellular proliferation could amplify the background mutation rate. This mutational amplification increased tumor formation in experimental animals.^37

–40 Studies conducted by Moolgavkar and Knudson also supported an important role for cellular proliferation during this era.⁴¹ Throughout the 1990s, Ames and Gold incorporated these new findings on proliferation-induced amplification of background mutation rate into their thinking resulting in a series of publications, one of which we highlighted in Smith and Perfetti,³⁵ that is, Ames and Gold.⁴² Published in Science, Ames and Gold,⁴² was actually a commentary on the Cohen and Ellwein paper published in the same issue.³⁸ In 2017, Tomasetti and Vogelstein provided evidence for the clinical relevance in humans of the Ames and Gold mechanism regarding the amplification of the background mutation rate.⁴³ These authors sought to determine the relative contribution to human cancers from inherited mutations, mutations induced by environmental factors, or mutations resulting from DNA replication errors (R). They compared the number of normal stem cell divisions with the risk of 17 cancer types occurring in 69 different countries. Tomasetti and Vogelstein ⁴³ reported the following results:

The data revealed a strong correlation (median ¼ 0.80) between cancer incidence and normal stem cell divisions in all countries, regardless of their environment. The major role of R mutations in cancer etiology was supported by an independent approach, based solely on cancer genome sequencing and epidemiological data, which suggested that R mutations are responsible for two-thirds of the mutations in human cancers.

In summary, accumulated mutations due to DNA replication are the driving force behind the majority of human cancers, that is, mitogenesis increases mutagenesis in humans as well as in rodents. The conclusions of Tomasetti and Vogelstein⁴³ were consistent with those expressed by Armitage and Doll.⁴⁴ Both Armitage and Doll⁴⁴ and Tomasetti and Vogelstein⁴³ held stem cell number and proliferation rates constant. However, both stem cell number and proliferation rate can be influenced by environmental factors. A broader model incorporating potential environmental influences on cell number, proliferation rates, and mutation rates (secondary to DNA reactive carcinogens) formed the basis for the Cohen and the Moolgavkar models.

Stages of malignant tumor development

There are several separate but interrelated conceptual frameworks for ideating the life cycle of cancer from its origins as a single normal cell to its final stage as a large mass of billions of locally invasive or metastatic cells within a malignant tumor. One of the most useful conceptual frameworks has been the two-stage mouse dermal promotion assay from which the terms initiation, promotion, and progression derive.^45,46 The eponymous term “initiation” refers to the first step in the mouse dermal promotion assay.⁴⁷ A parent chemical that reacts with DNA can serve as an initiator, although metabolic enzymes are frequently required to convert a nonreactive parent chemical into an electrophilic mutagen.⁴⁸ Different tissues and different species can possess significantly different levels of metabolic enzyme activity. This differential in metabolic potential confers tissue and species specificity on many initiators.⁴⁹

It is important to understand that the effects of initiators are irreversible. After the DNA in a particular cell has been altered by a chemical initiating agent, that cell is susceptible to promotion until its death. Daughter cells produced from the division of the mutated cell will also carry the mutation.⁴⁸ A linear relationship has been observed between the dose of initiator and the quantity of tumors produced in studies of mouse skin carcinogenesis. Therefore, any exposure to an initiator increases cancer risk and this risk increases with higher levels of exposure.⁴⁹

Promotion refers to the process of enhancing cellular proliferation.⁵⁰ Cells mutated by an initiator are susceptible to the effects of promoters. Promotion gives rise to a large number of daughter cells containing the original mutation induced by the initiating chemical.⁵¹ Promoters do not affect cells not previously treated with an initiator.⁴⁹ Unlike initiators, promoters do not covalently bind to DNA or cellular macromolecules. Promoters can be categorized as specific or nonspecific. Specific promoters interact with receptors on or in target cells of defined tissues. Nonspecific promoters alter gene expression without binding to a known receptor.⁴⁸ Tissue and species specificity results from the differential distribution of receptors on various tissue types. The effect on tumor growth of promoter application is dose-dependent. However, promotion displays both threshold and ceiling effects with both a minimum dose required and a maximum dose beyond which no further effects are seen.⁴⁹

Progression refers to the stepwise transformation of a benign tumor to a malignant tumor. Repeated application of a promoting agent onto previously initiator-treated mouse skin can produce benign papillomas. After cessation of treatment with promoter, most of these papillomas regress but some progress to cancer.⁵² Benign papillomas that progress to cancer have acquired an additional, spontaneous mutation.⁵³ Progression is associated with karyotypic change as virtually all benign tumors that advance to malignant tumors become aneuploid (incorrect number of chromosomes). Karyotypic change is associated with increased growth rate, invasiveness, metastatic potential, and biochemical and morphological changes.⁴⁹

All of the major human carcinomas pass through histological stages of hyperplasia, dysplasia, carcinoma in situ, and invasive carcinoma.⁵⁴ Cancers of the central nervous system, bone, blood, and lymph system do not use the same terminology due to differences in pathology. In humans, molecular stages analogous to tumor initiation, promotion, and progression are concomitant with but variably overlapping with the sequential histological stages of carcinoma development, including hyperplasia (increase in cell number), dysplasia (precancerous stage), carcinoma in situ (not yet invasive or noninvasive carcinoma), invasive carcinoma, and metastatic carcinoma.

Angiogenesis is a late change in tumor development

Tumors can grow to approximately 1–2 mm³ before metabolic demands restrict growth due to the diffusion limit of oxygen and nutrients. Growth beyond this size requires the tumor to switch to an angiogenic phenotype.⁵⁵ Angiogenesis is a multistep process by which new blood vessels sprout from preexisting capillaries. During angiogenesis, activated endothelial cells proliferate and migrate to specific target locations and assemble into new capillary tubes.⁵⁶ A vascular lumen is constructed by the synthesis of a new basement membrane and maturation of vessels. Vasculogenesis differs from angiogenesis as it involves de novo differentiation of endothelial cells from in situ mesoderm-derived precursor cells. The principal process in angiogenesis is sprouting from preexisting blood vessels. Under physiological and pathological conditions, a secondary contribution to angiogenesis includes recruitment and differentiation in situ of bone marrow–derived endothelial progenitor cells.⁵⁷ Angiogenesis plays a crucial role in several normal and pathological processes including embryogenesis, the female reproductive cycle, tissue repair, wound healing, cardiac ischemia, limb ischemia, neovascularization associated with diabetic retinopathy, rheumatoid arthritis, and neoplasms.⁵⁸ Angiogenic and antiangiogenic factors are secreted by both tumor cells and host cells infiltrating tumors and interact to influence the growth and progression of cancer.⁵⁶ As tumors grow, they need additional oxygen and nutrients. In the absence of a blood supply from angiogenesis, the growing tumor can become latent or its cells can die.⁵⁹ A number of angiogenic growth factors including angiogenin, angiopoietin-1, cyclo-oxygenase, hepatocyte growth factor, and tumor growth factor are produced by tumor cells or host cells. In contrast with the angiogenic growth factors, antiangiogenic factors including angiopoietin-2, angiostatin, interferon-α, interferon-β, endostatin, and vasostatin hold the existing blood vessels in a quiescent state.⁶⁰ Hypoxia regulates the vascularity of the tumor via different cells in the tumor microenvironment including vascular endothelial cells, pericytes, and bone marrow precursor cells.^61,62

In the life cycle of a malignant carcinoma, angiogenesis is a relatively late event that frequently occurs decades following the initial exposure to mutagens.⁶³ Table 1 presents evidence that small previously avascular tumors enter a rapid growth phase following vascularization via angiogenesis. In contrast with the decades long time period of the overall carcinogenic process, this final phase of tumor progression appears to occur on a much more limited timeframe of a few to several years (Table 1). The extremely long lag phase between exposure to different chemical carcinogens and the clinical presentation of cancer (Table 2) supports the late introduction of the angiogenic process and its attendant exponential increase in tumor growth.⁸¹ For example, the average age of presentation for asbestos-related mesothelioma is 69 years,⁸² and the average age for lung cancer presentation in cigarette smokers is approximately 70 (Table 2).⁸³

Table 1.

Relative brevity of vascularized progression phase of squamous cell lung cancer resulting in treatment failure.

Typical age at initial exposure to mutagens/carcinogens^a	Typical age when sequential mutations transform a single cell^b	Typical age when squamous cell carcinoma reaches 10–30 mm^c (stage III/IV)^d	Time in progression phase after presentation until death—with treatment^e	Time in progression phase after presentation until death—without treatment (months)	Five-year survival rate after presentation at stage III or IV (%)	References
18–25	40–70	45–75	6–24 months (IV)	2–6	1–15	Birring and Peake⁶⁴; Hu et al.⁶⁵; Yuan et al.⁶⁶
18–25	40–70	45–75	Up to 5 years (III–IV)	2–6	5–15	Lindell et al.⁶⁷
18–25	40–70	45–75	Up to 3 years (III–IV)	2–6	<10	Geddes⁶⁸
18–25	40–70	45–75	Not specified	2–6	1–15	Pedersen et al.⁶⁹
18–25	40–70	45–75	Study on-going	2–6	1–15%	Wille et al.⁷⁰
18–25	40–70	45–75	Up to 3 years (III–IV)	3–6	1–10	Detterbeck and Gibson⁷¹
18–25	40–70	45–75	∼18 months	<10	1–10	Lauro et al.⁷²

^aCigarette smoking usually initiates in late teens to early 20s. Occupational exposures usually begin following graduation from high school and into the early to mid-20s.

^bLung cancer diagnosis is rare prior to age 40. The average age at clinical presentation is approximately 70, meaning that half of all cases occur before age 70 and half after age 70.

^cIt takes approximately 8 years for a single squamous cell carcinoma to expand to approximately 30 mm. The growth rate of cancer cells is exponential. The natural history of malignant nodules as small as 5 mm detected on computed tomography scans is not well described and even nodules only 10 mm in size containing 10⁹ cells represent a fairly late stage in the disease process, considering that at death lung cancer tumors typically have 10¹² cells (Birring and Peake).⁶⁴

^dThe time from malignant transformation of a single cell to the formation of a small avascular or poorly vascularized tumor 10–30 mm is not well understood but the growth rate is believed to be relatively slow (approximately 8 years) prior to vascularization via angiogenesis.

^eThis vascularized tumor is in its exponential growth phase.

Table 2.

Average age of pulmonary cancer diagnosis.

Cancer type	Latency	Average age of lung cancer diagnosis	Reasons	Male/female	Smokers/nonsmokers (S/N)	References
All forms of lung cancer	54.48	69	Indoor air pollution	Male	Not specified	Hu et al.⁶⁵; Boulanger et al.⁷³
All forms of lung cancer	56.17	69	Smoking	Both	Not specified	Hu et al.⁶⁵; Yuan et al.⁶⁶
All forms of lung cancer	19	56	Uranium/Radon	Male	Both	Archer et al.⁷⁴; Jones⁷⁵
All forms of lung cancer	25	60	Uranium/Radon	Male	Both	Archer et al.⁷⁴; Jones⁷⁵
Lung cancer	—	69	Radon	Both	Not specified	Boulanger et al.⁷³
Lung cancer	—	69	Environmental Tobacco Smoke (ETS)	Both	Not specified	Boulanger et al.⁷³
Lung cancer	20	69.5	Bis(chloromethyl) ether	Both	Not specified	Gowers et al.⁷⁶
Mesothelioma	20	69	Asbestos-related (all types)	Both	Not specified	Howard⁷⁷; Anonymous⁷⁸
Mesothelioma	22.8	69	Asbestos-related (all types)	Both	Not specified	Kim et al.⁷⁹; Anonymous⁷⁸
Mesothelioma	22.2	69	Asbestos-related (all types)	Male	Not specified	Frost⁸⁰; Anonymous⁷⁸
Mesothelioma	28.2	69	Asbestos-related (all types)	Female	Not specified	Frost⁸⁰; Anonymous⁷⁸

Inflammation is associated with hypoxia

Pulmonary inflammation can result from a wide variety of agents and actions causing injury to the cells lining the airways, including inhalation of a wide variety of chemicals, including irritants, cytotoxic compounds, and certain metals and metallic complexes.³¹ The vast majority of neutrophils and macrophages found in pulmonary inflammation are not normally resident in the lung, but rather are recruited to inflammatory lesions.⁸⁴ One of the metabolic changes associated with active inflammation is development of hypoxia with concomitant accumulation of lactic acid and sometimes metabolic acidosis depending on the amount of lactic acid and the degree of buffering in the tissue environment.^85
–87 A number of factors can contribute to inflammation-induced tissue hypoxia including increased metabolic demands of cells and reductions in metabolic substrates caused by thrombosis (blood clots), trauma, compression (interstitial hypertension), or atelectasis (airway plugging).⁸⁸

Hypoxia due to inflammation is a stressor that can preferentially select for expansion of clonal populations of cells with particular mutations, for example, preferential survival of epidermal growth factor receptor (EGFR) positive clones.⁸⁹ In hypoxic mice with lung tumors induced by urethane, significant overexpression of EGFR, fibroblast growth factor receptor 2, and platelet-derived growth factor receptor were seen.⁸⁹ Similarly, using the HCC827 NSCLC cell line, Lu et al.⁹⁰ provided evidence that hypoxia/hypoxia-inducible factor (HIF)-2α activation mediates upregulation of EGFR protein levels. This observation provides a potential non-mutational explanation for the EGFR overexpression often seen in human adenocarcinomas. The authors hypothesized that “The data presented in this contribution also introduce the intriguing possibility that the tumor microenvironment may act as a universal oncogenic trigger that drives the autonomous growth of tumor cells.”

Human epidermal growth factor (EGF) is a 6 kilodalton protein and cytokine^91,92 comprised of 53 amino acid residues and containing three intramolecular disulfide bonds.⁹³ EGFR is a protein found on the surface of some cells and to which EGF binds. Cell division is stimulated following the binding of EGF to EGFR. EGFR is found at abnormally high levels on the surface of many types of cancer cells, so these cells may divide excessively in the presence of EGF. Alternate names for EGFR are ErbB1 and HER1.⁹⁴

Non-small cell lung cancers (NSCLs) are a group of lung cancers so named for either the normal cell of origin or the microscopic appearance of the tumor cells. The three main types of NSCL are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. NSCL is the most common kind of lung cancer.⁹⁵ Approximately 10% of patients with NSCLC in the United States and 35% in East Asia have a mutation in the EGFR gene in the DNA of their lung tumor.^96
–98 In both the United States and East Asia, EGFR mutations are more common in tumors from female never smokers (<100 cigarettes in patient’s lifetime) with adenocarcinoma histology.^96
–98 The percentage of EGFR mutation frequency rises to 60–65% in female East Asian never-smoker adenocarcinoma patients.⁹⁹ However, EGFR mutations are sometimes seen in squamous cell carcinoma and large cell carcinoma in both former and current smokers.¹⁰⁰

The EGFR mutations occur with exons 18–21. (Both the DNA sequence within a gene and the corresponding sequence in RNA transcripts are termed exons.¹⁰¹) EGFR exons 18–21 encode a portion of the EGFR kinase domain. EGFR mutations are usually heterozygous and display gene amplification, that is, increase in number of copies.¹⁰² The overwhelming majority, that is, approximately 90% of EGFR mutations, are deletions in exon 19 or point mutations in exon 21 L858R.¹⁰³ These mutations increase the kinase activity of EGFR. Kinase-induced phosphorylation activates signaling pathways that block apoptosis.¹⁰⁴ In the vast majority of cases, EGFR mutations seen in NSCLC do not overlap with KRAS mutations observed in the same tumors,¹⁰⁰ although KRAS mutations are found in 25–35% of newly diagnosed non-small cell, non-squamous cell patients.⁹⁹

Overexpression of HIF-1α and HIF-2α is common in human cancers

HIF-1 is a dimeric protein complex that plays an important role in the body’s response to hypoxia. HIF-1 is a transcription factor for dozens of target genes. The best-known function of HIF-1 is increasing vascularization in hypoxic tissues caused by ischemia or tumors.¹⁰⁵ Other functions of HIF-1 include immunological responses, physiological regulation of homeostasis, vascularization, and anaerobic metabolism. Due to its angiogenic properties, HIF-1 facilitates the survival and proliferation of cancer cells.¹⁰⁵

In well-developed human tumors, expression of HIF-1α and HIF-2α is common (Table 3). Over 70% of all human cancers are HIF positive.^107,108 A majority of solid tumors, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas,¹⁰⁹ reported nuclear expression of HIF-1α and HIF-2α in subsets of the different tumor cells. As compared with the relevant normal tissues, HIF-1α was overexpressed in 13 of 19 histological tumor types including colon, breast, gastric, lung, skin, ovarian, pancreatic, prostate, and renal carcinomas. Abnormal p53 accumulation and cellular proliferation was associated with HIF-1α expression. Dysplastic lesions in breast, colon, and prostate tissue overexpressed HIF-1α, while benign tumors of the breast and uterus did not. Only 29% of primary breast cancers displayed HIF-1α expression. In contrast, 69% of breast cancer metastases showed HIF-1α expression. In brain tumors, areas of the brain vascularized by angiogenesis stained positive for the presence of HIF-1α. All of these data support an important role for HIF-1α in human cancer progression, that is, the final stage of the malignant tumor development process.¹⁰⁶

Table 3.

HIF-1α protein expression in human tumors and their metastases.^a

Tumor types	N ^b	−	+	++	+++	++++
Malignant primary tumors
Prostate adenocarcinoma	11	2	5	1	3
Colon adenocarcinoma	22		1	4	6	11
Breast adenocarcinoma	52	37	5	4	1	5
Lung adenocarcinoma	2					2
Lung small cell carcinoma	1					1
Renal clear cell carcinoma	1			1
Pancreas carcinoma	5	1	2	2
Ovarian carcinoma	2			1	1
Gastric carcinoma	2		1			1
Brain tumors	9	4	2			3
Mesothelioma	1				1
Melanoma	4		3	1
Malignant fibrous histiocytoma	1		1
Hepatocellular carcinoma	8	8
Thyroid carcinoma	2	2
Lymphoma	3	3
Rhabdomyosarcoma	1	1
Epithelioid sarcoma	1	1
Carcinoid	3	3
Malignant tumors in total	131	62	20	14	12	23
Metastatic tumors
Lymph node metastases from:
Prostate adenocarcinoma	1	1
Breast adenocarcinoma	13	4	5		2	2
Colon adenocarcinoma	10	1	1	1	5	2
Bone metastases from:
Prostate adenocarcinoma	10	6	2	1	1
Vena caval invasion from:
Renal clear cell carcinoma	1					1
Undifferentiated carcinoma	1					1
Metastatic tumors in total	36	12	8	2	8	6
Benign tumors
Breast fibroadenoma	10	10
Uterine leiomyoma	2	2
Benign tumors in total	12	12

HIF: hypoxia-inducible factor.

^aAdapted from Zhong et al.¹⁰⁶

^b N, number of cases analyzed; −, no staining; +, nuclear staining in less than 1% of cells; ++, in 1–10% of cells; +++, in 10–50%; ++++, greater than 50%.

Additional lines of evidence support a role for HIF-1α in the later and final stages of malignant tumor progression. HIF-1α overexpression seen in biopsies of brain, breast, cervical, esophageal, oropharyngeal, and ovarian cancers is positively associated with treatment failure and patient mortality.¹¹⁰ In human breast and brain cancers, HIF-1α overexpression strongly correlates with tumor grade (degree of microscopic abnormality) and vascularity.¹¹¹ In metastatic osteosarcoma, HIF-1α impacts tumorigenesis, tumor metabolism, differentiation, angiogenesis, proliferation, metastasis, and ultimately patient survival.^112,113 HIF-1α is overexpressed in gastrointestinal stroma of stomach tumors and in gastric adenocarcinomas but absent in normal gastric mucosa.¹¹⁴

Transformed cell lines are not analogous to pre-initiated cells

Before summarizing the data on the effect of cobalt on HIF-1 expression in cultured cells, it is necessary to understand the many differences between immortalized cell lines and primary cell cultures. The genetic characteristics of transformed cells include aneuploidy, heteroploidy, high spontaneous mutation rate, overexpressed oncogenes, and mutated or deleted suppressor genes. Structural changes of transformed cells include altered cytoskeleton, changed extracellular matrix, modified expression of cell adhesion molecules, and disrupted cell polarity. Growth characteristics of transformed cells include immortalization, loss of contact inhibition, anchorage independence, reduction in growth density limits, growth factor independence, low serum requirement, and a shorter population doubling time. Neoplastic characteristics of transformed cells include tumorigenicity, invasiveness, and increased protease secretion. In summary, transformation is associated with genetic instability, immortalization, aberrant growth control, and malignancy.¹¹⁵

Cobalt can mimic hypoxia in vitro

A number of different in vitro studies have exposed primary and transformed cells to cobalt compounds and measured expression of HIF-1. Table 4 lists the cobalt source, cell lines; whether the cell line was HIF-1 positive following cobalt compound administration; and the reference. Cobalt is an essential element in cellular growth and development. The level of cobalt in cells is incredibly small (in the ppb range). In both primary and immortalized cell lines, very small amounts of cobalt is added in the growing medium.¹²⁶ Both primary and immortalized cell lines exposed to cobalt (a hypoxia mimetic agent) will induce the accumulation of HIF-1α protein in primary cell cultures under normal oxygen tension (normoxia).^127–128 The concentration of Co ions is somewhat elevated for induction of overexpression of HIF-1 to occur (>50 µM) under normoxia.

Table 4.

Cobalt as a hypoxia mimetic in primary and transformed cell lines.

Cobalt source	Cells	HIF positive (Y/N)	References
CoCl₂	Human mesenchymal stem cells (dental pulp, umbilical cord, and adipose tissue; Primary)	Y (variable by MSC type)	Teti et al.¹¹⁶
CoCl₂	Airway smooth muscle cells (Primary)	Y	Chachami et al.¹¹⁷
CoCl₂	Human dermal microvascular endothelial cell line (Primary), human pulmonary aortic endothelial cells (Primary), and human pulmonary microvascular endothelial cell line (Primary)	Y	Kim et al.¹¹⁸
CoCl₂	Human umbilical vein endothelial cells (Primary)	Y	Liu et al.¹¹⁹
CoCl₂	Umbilical cord blood-derived CD133⁺ cells (Primary)	Y	Zan et al.¹²⁰
CoCl₂	Umbilical vein endothelial cells and skin fibroblasts (Primary)	Y	Vissers et al.¹²¹
CoCl₂	U251 human glioblastoma cell line (Transformed)	Y	Al Okail¹²²
CoCl₂	Ciliated MDCK primary cell culture	Y	Resnick¹²³
CoCl₂	Primary breast cell lines (HCC1395 and HCC1937) and metastatic primary breast cell lines (MCF-7 and MDA-MB-231; Transformed)	Y	Fu et al.¹²⁴
CoCl₂	A549 human lung adenocarcinoma cells (Transformed)	Y	Uchida et al.¹²⁵

CoCl₂: cobalt chloride; HIF: hypoxia-inducible factor.

Conclusions

Chronic NTP rodent inhalation studies using cobalt sulfate and cobalt metal report an increase in lung and other tumors in rats.^1,2 Previously, our group analyzed the entire NTP database^31,35,36 and supported the original conclusions of Cohen and colleagues,^37

–40 Moolgavkar and Knudson,⁴¹ and Ames and Gold ⁴² regarding an important role for increased cellular proliferation rates resulting from cytotoxicity induced by high doses of test agent. The increased cellular proliferation rates amplify background mutation rates and induce tumors in rodents not related to genotoxic effects. NTP does not consider the role of mitogenesis inducing mutagenesis as an explanation underlying the formation of rat pulmonary tumors despite the pro-inflammatory effect of inhaled cobalt compounds. In contrast, one of the mechanisms invoked to explain the increase in rat lung tumors in the NTP study, and why that increase would be relevant to humans, is an increase in HIF-1 from cobalt exposure. Rodent pulmonary tumors differ from human lung cancers in that the human tumors tend to be highly vascularized thereby possessing metastatic potential. In contrast, rodent pulmonary tumors tend to be localized and less vascularized. Inhalation of cobalt compounds in rats and mice does not model the late final progression stage of human pulmonary carcinogenesis but rather attempts to model the earlier stages of tumor initiation and promotion not affected by overexpression of HIF.³⁶

Several lines of evidence suggest that in vitro demonstrations of cobalt-induced increases in HIF-1 are not relevant to human cancers. The mechanism by which overexpression of HIF-1 would be expected to influence cancer would be an increased tendency toward angiogenesis thereby accelerating the later progression stages of the disease. The very large number of human patients with long-term exposure to cobalt ions via leaching of MOM prosthetic implants who do not demonstrate an increased risk of cancer does not support a role for enhanced angiogenesis. Similarly, goats ingest up to 667 times the human ingestion level of cobalt throughout their lifetime of 15–18 years and demonstrate low cancer rates, that is, about a 1% incidence rate. If cobalt was inducing a clinically significant angiogenic response, the large exposures and significant life spans of goats might be expected to show at least some carcinogenic effect. In addition, 70% of all human cancers, including lung cancers, demonstrate overexpression of HIF-1 in the absence of cobalt exposure. Although cobalt can function as a hypoxia mimetic in cell culture studies, hypoxia routinely develops when tumors exceed the size at which oxygen and waste products can be exchanged via diffusion in the absence of cobalt^129

–132 rendering cobalt’s hypoxia mimetic properties redundant. In summary, the current weight of the evidence suggests that in vitro cobalt-stimulated HIF-1 overexpression does not correlate with cancer risk from cobalt exposure in humans.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Carr J Smith

References

National Toxicology Program (NTP). Toxicology and carcinogenesis studies of cobalt sulfate heptahydrate (CAS no. 10026-24-1) in F344/N rats and B6C3F₁ mice (Inhalation Studies). Technical Report Series No. 471. NIH Publication No. 98-3961. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC, https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr471.pdf (1998, accessed 2 February 2019).

National Toxicology Program (NTP). Toxicology and carcinogenesis studies of cobalt metal (CAS no. 7440-48-4) in F344/N rats and B6C3F1 mice (Inhalation Studies). Technical Report Series No. 581. NIH Publication No. 14-5923. U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC, https://ntp.niehs.nih.gov/ntp/about_ntp/trpanel/2013/october/draft_tr-581.pdf#search=cobalt%20metal (2013, accessed 2 February 2019).

National Toxicology Program (NTP). Final Report on Carcinogens (RoC) background document for cobalt sulfate. U.S. Department of Health and Human Services, Public Health Service, Research Triangle Park, NC, Contract Number N01-ES-85421, https://ntp.niehs.nih.gov/ntp/newhomeroc/roc11/coso4pub_no_appendices_508.pdf (2002, accessed 2 February 2019).

National Toxicology Program (NTP). Report on Carcinogens (RoC) background document for cobalt–tungsten carbide: powders and hard metals. U.S. Department of Health and Human Services, Public Health Service, https://ntp.niehs.nih.gov/ntp/roc/twelfth/2010/finalbds/hardmetalsbd20100408_508.pdf (2009, accessed 2 February 2019).

National Toxicology Program (NTP). Report on Carcinogens (RoC) monograph on cobalt and cobalt compounds that release cobalt ions in vivo. U.S. Department of Health and Human Services, Public Health Service, https://ntp.niehs.nih.gov/ntp/roc/monographs/cobalt_final_508.pdf (2016, accessed 2 February 2019).

Mur

Moulin

Charruyer-Seinerra

. A cohort mortality study among cobalt and sodium workers in an electrochemical plant. Am J Ind Med 1987; 11(1): 75–81.

Hogstedt

Alexandersson

. Mortality among hard metal workers [in German, English summary]. Arbete och Hälsa 1990; 21: 1–26.

Moulin

Wild

Mur

. A mortality study of cobalt production workers: an extension of the follow-up. Am J Ind Med 1993; 23(2): 281–288.

Lasfargues

Wild

Moulin

. Lung cancer mortality in a French cohort of hard-metal workers. Am J Ind Med 1994; 26(5): 585–595.

10.

Ozlü

Bülbül

Smoking and lung cancer. Tuberk Toraks 2005; 53(2): 200–209.

11.

Flanders

Lally

Zhu

. Lung cancer mortality in relation to age, duration of smoking, and daily cigarette consumption. Cancer Res 2003; 63(19): 6556–6562.

12.

Campbell

Esley

. Metal release from hip prostheses: cobalt and chromium toxicity and the role of the clinical laboratory. Clin Chem Lab Med 2013; 51(1): 213–220.

13.

Steinberg

Shah

Gartland

. Effects of chronic cobalt and chromium exposure after metal-on-metal hip resurfacing: an epigenome-wide association pilot study. J Orthop Res 2017; 35(10): 2323–2328.

14.

Hunt

Whitehouse

Beswick

. Implications of introducing new technology: comparative survivorship modeling of metal-on-metal hip replacements and contemporary alternatives in the national joint registry. J Bone Joint Surg Am 2018; 100(3): 189–196.

15.

Mäkelä

Visuri

Pulkkinen

. Cancer incidence and cause-specific mortality in patients with metal-on-metal hip replacements in Finland. Acta Orthop 2014; 85(1): 32–38.

16.

Mäkelä

Visuri

Pulkkinen

. Risk of cancer with metal-on-metal hip replacements: population-based study. BMJ 2012; 345: e4646.

17.

Smith

Dieppe

Porter

. Risk of cancer in first seven years after metal-on-metal hip replacement compared with other bearings and general population: linkage study between the National Joint Registry of England and Wales and hospital episode statistics. BMJ 2012; 344: e2383.

18.

Järvholm

Lewold

Malchau

. Age, bodyweight, smoking habits and the risk of severe osteoarthritis in the hip and knee in men. Eur J Epidemiol 2005; 20(6): 537–542.

19.

Gillispie

Henry

O’Connell

. Development of hematopoietic cancers after implantation of total joint replacement. Clin Orthop Relat Res 1996; (329 Suppl): S290–S296.

20.

Visuri

Pukkala

Paavolainen

. Cancer risk after metal on metal and polyethylene on metal total hip arthroplasty. Clin Orthop Relat Res 1996; (329 Suppl): S280–S289.

21.

Ensminger

Olentine

Heinemann

. Feeds and nutrition. 2nd ed. Upper Saddle River: Prentice Hall, 1989, pp. 1–1544, http://www.kaeco.com/nutritional-supplementation-for-goats/ (accessed 4 February 2019).

22.

Rashid

. Goats and their nutrition, Manitoba Goat Association. Manitoba Agriculture, Food and Rural Initiatives. 2008. https://www.gov.mb.ca/agriculture/livestock/goat/pubs/goats-and-their-nutrition.pdf (accessed 30 January 2019).

23.

Nordberg

Fowler

Nordberg

. Handbook on the toxicology of metals. New York: Academic Press, 2014, pp. 1–1542.

24.

Abegglen

Caulin

Chan

. Potential mechanisms for cancer resistance in elephants and comparative cellular response to DNA damage in humans. JAMA 2015; 314(17): 1850–1860.

25.

Lohr

. One hundred two tumors in 100 goats (1987–2011). Vet Pathol 2012; 50(4): 668–675.

26.

Versnaeyen

Kolkman

Van Aert

. Multicentric B-cell lymphoma in a pygmy goat. Vlaams Diergeneeskundig Tijdschrift 2016; 85(5): 291–295.

27.

Zahm

Blair

. Pesticides and non-Hodgkin’s lymphoma. Cancer Res. 1992; 52(19 Suppl): 5485s–5488s.

28.

Spector

. Handbook of Biological Data. In: Spector

(ed) Philadelphia: W.B. Saunders, 1956, p. 584. ASIN: B000F2K6TS.

29.

Nolte

. Teeth, life expectancy and how to estimate a goat’s age, goat health and husbandry, http://fiascofarm.com/goats/age.htm (2017, accessed 2 February 2019).

30.

Smith

Perfetti

. Tumor site concordance and genetic toxicology test correlations in NTP 2-year feed studies. Toxicol Res Appl 2017; 1: 1–12.

31.

Smith

Anderson

. High discordance in development and organ site distribution of tumors in rats and mice in NTP 2-year inhalation studies. Toxicol Res App 2017; 1: 12–22.

32.

Smith

Perfetti

. Tumor site concordance and genetic toxicology test correlations in NTP two-year gavage, drinking water, dermal, and intraperitoneal injection studies. Toxicol Res App 2018a; 2: 1–18.

33.

Smith

Perfetti

. Ames mutagenicity, structural alerts of carcinogenicity, Hansch QSAR parameters (ClogP, CMR, MgVol), tumor site concordance/multiplicity, and tumorigenicity rank in NTP 2-year rodent studies. Toxicol Res App 2018; 2: 1–14.

34.

Smith

Perfetti

. Comparison of carcinogenicity predictions by the oncologic expert system, and National Center for Toxicological Research liver cancer database (NCTRlcdb) with NTP two-year rodent study tumorigenicity results. Toxicol Res App 2018b; 2: 1–11.

35.

Smith

Perfetti

. The ‘false positive’ conundrum in the NTP 2-year rodent cancer study database. Toxicol Res App 2018; 2: 1–13.

36.

Smith

Perfetti

King

. Bronchioloalveolar lung tumors induced in “mice only” by non-genotoxic chemicals are not useful for quantitative assessment of pulmonary adenocarcinoma risk in humans. Toxicol Res App 2018; 2: 1–24.

37.

Greenfield

Ellwein

Cohen

. A general probabilistic model of carcinogenesis: analysis of experimental urinary bladder cancer. Carcinogenesis 1984; 5(4): 437–445.

38.

Cohen

Ellwein

. Cell proliferation in carcinogenesis. Science 1990; 249(4972): 1007–1011.

39.

Cohen

Ellwein

. Genetic errors, cell proliferation, and carcinogenesis. Cancer Res 1991; 51(24): 6493–6505.

40.

Cohen

Purtillo

Ellwein

. Ideas in pathology. Pivotal role of increased cell proliferation in human carcinogenesis. Mod Pathol 1991; 4(3): 371–382.

41.

Moolgavkar

Knudson

Jr . Mutation and cancer: a model for human carcinogenesis. J Natl Cancer Inst 1981; 66(6): 1037–1052.

42.

Ames

Gold

. Too many rodent carcinogens: mitogenesis increases mutagenesis. Science 1990; 249(4972): 970–971.

43.

Tomasetti

Vogelstein

. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 2015; 347(6217): 78–81.

44.

Armitage

Doll

. The age distribution of cancer and a multi-stage theory of carcinogenesis. Br J Cancer 1954; 8(1): 1–12.

45.

Slaga

Argyris

. Promotion of carcinomas by repeated abrasion in initiated skin of mice. Cancer Res 1981; 41(12 Pt 1): 5193–5195.

46.

Walborg

DiGiovanni

Conti

. Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates. Toxicol Sci 1998; 45(2): 137–145.

47.

Anonymous. Initiation: cancer biology – cancer development, Emory Winship Cancer Institute, National Cancer Institute Designated Comprehensive Cancer Center, Cancer Quest, https://www.cancerquest.org/cancer-biology/cancer-development (2018, accessed 29 December 2018)

48.

Troll

Wiesner

. The role of oxygen radicals as a possible mechanism of tumor promotion. Annu Rev Pharmacol Toxicol 1985; 25: 509–528.

49.

Pitot

Goldsworthy

Moran

. The natural history of carcinogenesis: implications of experimental carcinogenesis in the genesis of human cancer. J Supramol Str Cell 2004; 17(2): 133–146.

50.

Anonymous. Promotion: cancer biology – cancer development, Emory Winship Cancer Institute, National Cancer Institute Designated Comprehensive Cancer Center, Cancer Quest, https://www.cancerquest.org/cancer-biology/cancer-development (2018, accessed 29 December 2018).

51.

Yamagiwa

Ichikawa

. Experimental study of the pathogenesis of carcinoma. J Cancer Res 1918; 3: 1–29.

52.

Anonymous. Progression: cancer biology – cancer development, Emory Winship Cancer Institute, National Cancer Institute Designated Comprehensive Cancer Center, Cancer Quest, https://www.cancerquest.org/cancer-biology/cancer-development (2018, accessed 29 December 2018).

53.

Alberts

Johnson

Lewis

. Molecular biology of the cell. 4th edition, Ch. 23, Cancer. New York: Garland Science, 2002.

54.

Anonymous. U.S. Department of Health and Human Services, National Institutes of Health, National Cancer Institute, understanding cancer, SEER training modules – cancer terms, https://training.seer.cancer.gov/disease/cancer/terms.html (2018, accessed 29 December 2018).

55.

Hillen

Griffioen

. Tumour vascularization: sprouting angiogenesis and beyond. Cancer Metastasis Rev 2007; 26(3-4): 489–502.

56.

Pang

Poon

. Clinical implications of angiogenesis in cancers. Vasc Health Risk Manag 2006; 2(2): 97–108.

57.

Asahara

Masuda

Takahashi

. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85(3): 221–228.

58.

Carmeliet

Jain

. Angiogenesis in cancer and other diseases. Nature 2000; 407(6801): 249–257.

59.

Hanahan

Weinberg

. Hallmarks of cancer: the next generation. Cell 2011; 144(5): 646–674.

60.

Ferrara

. The role of VEGF in the regulation of physiological and pathological angiogenesis. EXS 2005; 94: 209–231.

61.

Weis

Cheresh

. Tumor angiogenesis: molecular pathways and therapeutic targets. Nature Med 2011; 17(11): 1359–1370.

62.

Semenza

. Cancer-stromal cell interactions mediated by hypoxia-inducible factors promote angiogenesis, lymphangiogenesis, and metastasis. Oncogene 2013; 32(35): 4057–4063.

63.

Loeb

Harris

. Advances in chemical carcinogenesis: a historical review and prospective. Cancer Res 2008; 68(17): 6863–6872.

64.

Birring

Peake

. Symptoms and the early diagnosis of lung cancer. Thorax 2005; 60(4): 268–269.

65.

Liu

. [Estimation of latency period of lung cancer]. [Article in Chinese]. Zhonghua Zhong Liu Za Zhi 1994; 16(1): 18–21.

66.

Yuan

Cao

Rustam

. Time-to-progression of NSCLC from early to advanced stages: an analysis of data from SEER registry and a single institute. Sci Rep 2016; 6: Article number: 28477. https://doi.org/10.1038/srep28477

67.

Lindell

Hartman

Swensen

. Five-year lung cancer screening experience: CT appearance, growth rate, location, and histologic features of 61 lung cancers. Radiology 2007; 242(2): 555–562.

68.

Geddes

. The natural history of lung cancer: a review based on rates of tumour growth. Br J Dis Chest 1979; 73(1): 1–17.

69.

Pedersen

Ashraf

Dirksen

. The Danish randomized lung cancer CT screening trial--overall design and results of the prevalence round. J Thorac Oncol 2009; 4(5): 608–614.

70.

Wille

Dirksen

Ashraf

. Results of the randomized Danish lung cancer screening trial with focus on high-risk profiling. Am J Respir Crit Care Med 2016; 193(5): 542–551.

71.

Detterbeck

Gibson

. Turning gray: the natural history of lung cancer over time. J Thorac Oncol 2008; 3(7): 781–792.

72.

Lauro

Onesti

Righini

. The use of bevacizumab in non-small cell lung cancer: an update. Anticancer Res 2014; 34(4): 1537–1545.

73.

Boulanger

Bayeux

Mandin

. Socio-economic costs of indoor air pollution: a tentative estimation for some pollutants of health interest in France. Environ Int 2017; 104: 14–24.

74.

Archer

Coons

Saccomanno

. Latency and the lung cancer epidemic among United States uranium miners. Health Phys 2004; 87(5): 480–489.

75.

Jones

. What are the health costs of uranium mining? A case study of miners in Grants, New Mexico. Int J Occup Environ Health 2014; 20(4): 289–300.

76.

Gowers

DeFonso

Schaffer

. Incidence of respiratory cancer among workers exposed to chloromethyl-ethers. Am J Epidemiol 1993; 137(1): 31–42.

77.

Howard

, Minimum Latency & Types or Categories of Cancer. World Trade Center Health Program, 9.11 Monitoring and Treatment Program, https://www.cdc.gov/wtc/pdfs/policies/wtchpminlatcancer2014-11-07-508.pdf (2014, accessed 2 February 2019).

78.

Anonymous. Surveillance, Epidemiology, and End Results Program. Table 17.5. Mesothleioma survival rates by year of diagnosis. All races, males and females, https://seer.cancer.gov/archive/csr/1975_2006/results_merged/sect_17_mesothelioma.pdf (accessed 2 February 2019).

79.

Kim

Koh

Park

. Decision tree of occupational lung cancer using classification and regression analysis. Saf Health Work 2010; 1(2): 140–148.

80.

Frost

. The latency period of mesothelioma among a cohort of British asbestos workers (1978–2005). Brit J Cancer 2013; 109(7): 1965–1973.

81.

Weston

Harris

. Multistage carcinogenesis. In: Kufe

Pollock

Weichselbaum

. (eds.) Holland-frei cancer medicine. 6th ed. Hamilton: BC Decker, 2003. https://www.ncbi.nlm.nih.gov/books/NBK13982/ (accessed 31 January 2019).

82.

Selby

. Mesothelioma survival rates. The Mesothelioma center, https://www.asbestos.com/mesothelioma/survival-rate/ (2018, accessed 31 January 2019).

83.

Anonymous. Key statistics for lung cancer – American Cancer Society, https://www.cancer.org/cancer/non-small-cell-lung-cancer/about/key-statistics.html (2019, accessed 31 January 2019).

84.

Lewis

Lee

Underwood

. Macrophage responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol 1999; 66(6): 889–900.

85.

Kokura

Yoshida

Yoshikawa

. Anoxia/reoxygenation-induced leukocyte-endothelial cell interactions. Free Radic Biol Med 2002; 33(4): 427–432.

86.

Haddad

. Science review: redox and oxygen-sensitive transcription factors in the regulation of oxidant-mediated lung injury: role for hypoxia-inducible factor-1α. Crit Care 2003; 7(1): 47–54.

87.

Saadi

Wrenshall

Platt

. Regional manifestations and control of the immune system. FASEB J 2003; 16(8): 849–856.

88.

Eltzschig

Carmeliet

. Hypoxia and inflammation. N Engl J Med 2011; 364(7): 656–665.

89.

Karoor

Merrick

. Alveolar hypoxia promotes murine lung tumor growth through a VEGFR-2/EGFR dependent mechanism. Cancer Prev Res (Phila) 2012; 5(8): 1061–1071.

90.

Liu

Oeck

. Hypoxia promotes resistance to EGFR inhibition in NSCLC cells via the histone demethylases, LSD1 and PLU-1. Mol Cancer Res 2018; 16(10): 1458–1469.

91.

Harris

Chung

Coffey

. EGF receptor ligands. Exp Cell Res 2003; 284(1): 2–13.

92.

Yasuda

Park

Yun

. Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer. Sci Transl Med 2013; 5(216): 216ra177.

93.

Carpenter

Cohen

. Epidermal growth factor. J Biol Chem 1990; 265(14): 7709–7712.

94.

National Cancer Institute. NCI dictionary of cancer terms. https://www.cancer.gov/publications/dictionaries/cancer-terms/search?contains=false&q=epidermal+growth+factor (2018a, accessed 3 February 2019).

95.

National Cancer Institute. NCI dictionary of cancer terms. https://www.cancer.gov/publications/dictionaries/cancer-terms/def/non-small-cell-lung-cancer (2018b, accessed 3 February 2019).

96.

Lynch

Bell

Sordella

. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350(21): 2129–2139.

97.

Paez

Jänne

Lee

. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304(5676): 1497–1500.

98.

Pao

Miller

Zakowski

. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A 2004; 101(36): 13306–13311.

99.

Langer

. EGFR mutations in NSCLC. Targeted oncology. Published Online: Lung Cancer Case Studies, https://www.targetedonc.com/case-based-peer-perspectives/lung-cancer/langer-egfr-nsclc/egfr-mutations-in-nsclc (2018, accessed 3 February 2019).

100.

Lovly

Horn

Pao

. EGFR in non-small cell lung cancer (NSCLC). My Cancer Genome, https://www.mycancergenome.org/content/disease/lung-cancer/egfr/ (2015, accessed 3 February 2019).

101.

Biology-Online. Exon – Biology-Online Dictionary, https://www.biology-online.org/dictionary/ExonDec (2009, accessed 3 February 2019).

102.

Soh

Okumura

Lockwood

. Oncogene mutations copy number gains and mutant allele specific imbalance (MASI) frequently occur together in tumor cells. PLoS One 2009; 4(10): e7464.

103.

Ladanyi

Pao

. Lung adenocarcinoma: guiding EGFR-targeted therapy and beyond. Mod Pathol 2008; 21(Suppl 2): S16–S22.

104.

Sordella

Bell

Haber

. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 2004; 305(5687): 1163–1166. Epub ahead of print 29 July 2004.

105.

Ziello

Jovin

Huang

. Hypoxia-inducible factor (HIF)-1 regulatory pathway and its potential for therapeutic intervention in malignancy and ischemia. Yale J Biol Med 2007; 80(2): 51–60.

106.

Zhong

De Marzo

Laughner

. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases. Cancer Res 1999; 59(22): 5830–5835.

107.

Xia

Choi

Lee

. Recent advances in hypoxia-inducible factor (HIF)-1 inhibitors. Eur J Med Chem 2012; 49: 24–40.

108.

Masoud

. HIF-1α pathway: role, regulation and intervention for cancer therapy. Acta Pharm Sin B 2015; 5(5): 378–389.

109.

Talks

Turley

Gatter

. The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 2000; 157(2): 411–421.

110.

Semenza

. HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 2002; 8(4 Suppl): S62–S67.

111.

Semenza

Artemov

Bedi

. The metabolism of tumours: 70 years later. Novartis Found Symp 2001; 240: 251–260.

112.

Ouyang

. Hypoxia-inducible factor-1 expression predicts osteosarcoma patients’ survival: a meta-analysis. Int J Biol Markers. 2016; 31(3): e229–e234.

113.

Yang

. DEC2 expression is positively correlated with HIF-1 activation and the invasiveness of human osteosarcomas. J Exp Clin Cancer Res 2015; 34(1): 22.

114.

Rohwer

Lobitz

Daskalow

. HIF-1α determines the metastatic potential of gastric cancer cells. Br J Cancer 2009; 100(5): 772–781.

115.

Nandkishor

. Biology discussion. Cell transformation and characteristics of transformed cells, http://www.biologydiscussion.com/cell/cell-transformation/cell-transformation-and-characteristics-of-transformed-cells/10567 (2016, accessed 3 February 2019).

116.

Teti

Focaroli

Salvatore

. The hypoxia-mimetic agent cobalt chloride differently affects human mesenchymal stem cells in their chondrogenic potential. Stem Cells Int 2018; 2018: 24–40.

117.

Chachami

Simos

Hatziefthimiou

. Cobalt induces hypoxia-inducible factor-1 alpha expression in airway smooth muscle cells by a reactive oxygen species – and PI3K-dependent mechanism. Am J Respir Cell Mol Biol 2004; 31(5): 544–551.

118.

Kim

Rajagopal

Gonsalves

. A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells. J Immunol 2006; 177(10): 7211–7224.

119.

Liu

Wang

. Proteasomal degradation of O-GlcNAc transferase elevates hypoxia-induced vascular endothelial inflammatory response. Cardiovasc Res 2014; 103(1): 131–139.

120.

Zan

. Cobalt chloride enhances angiogenic potential of CD133⁺ cells. Front Biosci 2012; 17(4 Suppl): 2247–2258.

121.

Vissers

Gunningham

Morrison

. Modulation of hypoxia-inducible factor-1 alpha in cultured primary cells by intracellular ascorbate. Free Radic Biol Med 2007; 42(6): 765–772.

122.

Al Okail

. Cobalt chloride, a chemical inducer of hypoxia-inducible factor-1α in U251 human glioblastoma cell line. J Saudi Chem Soc 2010; 14(2): 197–201.

123.

Resnick

. HIF stabilization weakens primary cilia. PLoS One 2016; 11(11): e0165907.

124.

Hou

Yang

. Cobalt chloride-induced hypoxia modulates the invasive potential and matrix metalloproteinases of primary and metastatic breast cancer cells. Anticancer Res 2009; 29(8): 3131–3138.

125.

Uchida

Rossignol

Matthay

. Prolonged hypoxia differentially regulates hypoxia-inducible factor (HIF)-1 and HIF-2 expression in lung epithelial cells – implication of natural antisense HIF-1. J Biol Chem 2004; 279(15): 14871–14878.

126.

Mahey

Kumar

Arora

. Effect of cobalt(II) chloride hexahydrate on some human cancer cell lines. Springerplus 2016; 5(1): 930.

127.

Han

Xia

Song

. Comparative proteomic analysis of hypoxia-treated and untreated human leukemic U937 cells. Proteomics 2006; 6(11): 3262–3274.

128.

Dai

Gao

. Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells. J Exp Clin Cancer Res 2012; 31(1): 28.

129.

Rofstad

Galappathi

Mathiesen

. Fluctuating and diffusion-limited hypoxia in hypoxia-induced metastasis. Clin Cancer Res 2007; 13(7): 1971–1978.

130.

Hockel

Vaupel

. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001; 93(4): 266–276.

131.

Brown

Giaccia

. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 1998; 58(7): 1408–1416.

132.

Durand

Sham

. The lifetime of hypoxic human tumor cells. Int J Radiat Oncol Biol Phys 1998; 42(4): 711–715.