Hyperthermic Biology and Cancer Therapies: Title and subTitle BreakA Hypothesis for the “Lance Armstrong Effect”

There is perhaps no more important question in cancer research than to understand the molecular basis of how the majority of patients with testicular cancer can be treated so effectively. For instance, how could Lance Armstrong, who was diagnosed with very advanced metastatic testicular cancer, be treated so successfully that he could subsequently win multiple grueling Tours de France? Although such therapeutic success is now common for many patients with this type of advanced testicular cancer, this type of outcome is unattainable for the majority of patients with other types of advanced solid tumors. What accounts for the astounding therapeutic success with testicular cancer, and can this outcome be explained both at the cellular and molecular levels? Understanding the basis for the “Lance Armstrong effect” may provide therapeutic targets to enhance cures of other common advanced solid tumors that remain so refractory to the best systemic treatments.

The Lance Armstrong effect might primarily result from the unusual thermal sensitivity of normal testicular germ cells and their propensity to die when placed at the normal body temperature of 37°C. The metastatic testicular cancer cells that spread may retain this hyperthermic stress response to body temperature that would enhance their destruction through a more sensitive therapeutic-induced cell death to radiation or chemotherapy. Numerous clinical and basic studies have shown that hyperthermic stress can alter tumor cell kill and survival in a significant manner both in vivo and in vitro.¹ In addition, with many tumor types, hyperthermia (41°C-43°C) increases and synergizes the therapeutic response to combination therapy, such as radiation, cytotoxic drugs,² and immunotherapy.

Hyperthermia is a very old form of cancer therapy. There are more than 10 000 references listed in PubMed searches combining the terms of cancer and hyperthermia , but it still has not been widely accepted because of limitations in clinical application and understanding. With new types of thermal delivery systems, advances can be made for more precise delivery and better control. Molecular medicine techniques that deliver heat directly to specific tumor cells with nanoparticles have great potential. It is now possible to move from heating cancer cells externally to specifically heating the cancer cell internally. Specific tumor cell hyperthermia in combination with radiation, chemotherapy, and immunotherapy might provide a path to the therapeutic success realized by Lance Armstrong who had a tumor derived from a supersensitive, heat-responsive cell of origin.

Heat is an important microenvironmental and epigenetic factor in normal biological development. In some animals, sex is determined by environmental heat factors (epigenetics), whereas in other species, sex is determined by genetic factors through the presence of specific chromosomal signatures. The sex of amphibians is determined by the specific external temperature at which the egg is incubated within the nest. For example, alligator eggs located in the compostlike nature of the nest experience different localized temperatures during the time of their incubation. If the nest temperature is higher surrounding the egg, it hatches as a male. The opposite occurs if the egg incubation temperature is lower, with the hatchlets then being predominantly females.³ The opposite heat effect on sex determination is observed with turtles; elevated levels of heat during egg incubation produce predominantly female turtles, whereas lower temperatures produce male turtles. Once the heat-determined sex is realized, it is biologically irreversible and subsequent variances in heat do not alter the sex of the animal throughout its remaining life. In conclusion, in some animals incubation heat selects and imprints the sex expression in a critical period of time during early embryo development and the results are permanent.

In amphibians, the single egg is a stem cell that proceeds down 2 vastly different pathways of lifetime development (male or female) based solely on the heat level within the nest. In all animal species, a zygote is a single fertilized egg cell and as such serves as the primary totipotent stem cell for the development of all of the animal's cells. A recent report suggests that isolated adult testicular cells can be used as stem cells for cloning.⁴ Although this technique will only clone males, it may be a new model of study for stem cell research and avoids many of the ethical issues of using embryonic cells.

New insights into heat effects on egg stem cells, including molecular development and biology, may involve prime targets, such as DNA methylation, DNA rearrangements, noncoding RNAs, histone modification, and higher order DNA loop organization on the nuclear matrix.

Once sex is determined in all animals, either cold-blooded or warm-blooded, the embryo must be continuously incubated at a specific temperature throughout development to the time of hatching or birth. The second law of thermodynamics must be obeyed during all of these biological processes, including the development of the fetus with heat always flowing from high to low temperatures and still maintaining an overall increase in entropy for the entire system. The second law operates even during this period of increased biological complexity and self organization that is derived from heat.

Even in humans, ovulation is heralded in the female by an increase in basal body temperature that is of unknown significance. In contrast, male spermatogenesis ceases if the testes are subjected to a normal body temperature of 37°C, such as when the testes are inadvertently located within the abdomen and have not descended into the cooler scrotum that is their normal environment. The undescended testes are infertile and subject to an increased risk of developing cancer. The molecular mechanism of the effects of increased temperature on both human eggs and sperm formation and function needs to be resolved.

Warm-blooded animals evolved with normal temperatures that were often elevated far above the ambient temperature of their external environments. For both male and female animals to maintain this temperature differential is a very costly energy price for the organism. This vastly increased temperature expenditure must have a large survival benefit to have been positively selected so many times during evolution. The increased basal temperature and the ability to enhance it as fever during infection may be central to a biological defense of the host against pathogens. When mammals are infected with pathogens, they have adapted to produce increased body temperature (fever), which appears to be central to the proper function of the immune process and has been selected as a defense in animals over millions of years of evolution. Indeed, cold-blooded lizards are more prone to infection when the lizards are left in the cool shade, but when placed in the sun where their body temperature is increased, infected lizards survive.⁵ This benefit of body heat may have been enhanced in warm-blooded animal selection for survival in a pathogen-rich environment. The hyperthermia of fever may have been an adaptive but transient boost to immunity. The evolution of thermal biology has many complex features with many unresolved questions, including the anticancer effects it may possess. Fever temperatures are lowered to make individuals feel better but might be detrimental in fighting infections.⁶

There are many studies on the effects of heat on immunity. The antigenicity of molecules is often increased with temperature by unfolding the protein. Heat also stimulates the immune response through heat-induced immune cell functions and the transfer of the immune cells to infected sites.⁷ Understanding how heat increases immunity may be used to develop immune therapies.⁸ An additional example of the relationship between immune response and temperature is that sperm are actually antigenic to their host, although it is unknown whether the sensitivity of the testes to body temperature has an immunological component.

Cancer cells are more easily killed than normal cells if they are incubated above 42°C.⁹ The direct effect of heat on cultured cells and thermotolerance are complex events and involve rates and degrees of heating and rates of recovery and repair. Variability is often observed between cell types, thus making generalizations of heat effects difficult. However, the response of all cells to heat stress initiates the synthesis, distribution, and function of a family of proteins termed “heat shock proteins.”¹⁰ These proteins are involved in a wide variety of cellular functions and defenses, including dynamic protein folding, chaperoning functions, and transport throughout the cell. Heat shock proteins protect cellular elements from adversely increased temperatures. Specific heat shock proteins also appear as critical factors in cell signaling and have become prime factors as new therapeutic targets. Heat shock proteins should have a major role in understanding hyperthermia in cancer therapy. The role of heat shock proteins in apoptosis, mitochondrial activity, and nuclear function are developing areas of science.

One of the most sensitive cellular targets of hyperthermia is the nuclear matrix (Figure), a scaffold that organizes many functions within the nucleus. Studies of thermal effects on the nucleus by Roti Roti et al¹¹ and Lepock et al¹² have demonstrated thermally induced unfolding of the nuclear matrix and subsequent changes in the binding of specific proteins to the matrix. Variation (pleomorphism) and changes in nuclear structure are hallmarks of how cancer is diagnosed by the pathologist.¹³ These nuclear events are early neoplastic changes in all animal and human tumors and are the cellular basis of the diagnosis of all cancers regardless of the cause. The determination of nuclear structure is through a residual but dynamic structure of the nucleus termed the “nuclear matrix,” which is composed of only 10% of the total nuclear protein and provides a highly dynamic scaffold that is essentially free of lipids, histones, and nucleic acid.

In each cell, the nuclear matrix provides an anchoring site for more than 50 000 DNA loop domains.¹⁴ Each loop is approximately 60 000 bp in length and represents a replicon that is the units of DNA located between 2 replicating forks.¹⁴ These DNA replication sites are affixed to the matrix at the base of each DNA loop. During replication, the replicon loops are reeled down through these fixed replication sites located on the matrix. Near these sites of DNA replication machinery colocalized on the matrix are topoisomerases, steroid receptors, and high-mobility group proteins that are involved in chromosome translocations.¹⁵ The nuclear matrix provides many sites for higher-order DNA organization that organizes functional machinery for replication, transcription,¹⁶ and alternate splicing of the message. These loop domains are maintained in chromosome¹³ and sperm. The observations that the nuclear matrix and DNA loop organization are altered by hyperthermia could be of paramount importance in understanding how heat alters nuclear function.^{17 - 18}

Proteomic studies have revealed that the composition of the nuclear matrix is both tissue specific¹⁹ and tumor specific²⁰ and has been shown to be a source of novel cancer biomarkers.²¹ Thus, the studies by Roti Roti et al¹¹ and Lepock et al¹² of the nuclear matrix proteins as the most sensitive target for thermal response is of great potential importance in understanding the molecular mechanism of the Lance Armstrong effect.

Hyperthermia has been used alone and in combination with other forms of cancer therapy for many years but with only marginal clinical success. More recently, there have been a few clinical trials showing significant benefit of the addition of heat to radiation and chemotherapy.²² Some trials that have used more contemporary concepts in their design and analysis capitalized on eliminating the shortcomings in former hyperthermia logic. One advance has been that the thermal doses applied do not need to reach cytotoxic levels. More frequent application of lower thermal doses seem to result in tumor reoxygenation and reductions in DNA repair capacity of tumor cells, both critically important for enhanced tumor responses to radiation or chemotherapy.²³

Such hyperthermic therapy may be directed specifically to cancer cells while sparing the host cells. Magnetic iron particles have been directed to cancer cells by their inclusion into nanoparticles or liposomes.⁸ Once these micro iron particles in nanostructures are taken up by the tumors, they are heated by an external magnetic field set to frequencies that produce specific and controlled temperature within the tumor cells. Limitations in this approach might involve tumor volume, unwanted uptake by macrophages or fixed reticuloendothelial cells, or inadvertent lodging of the iron particles in unintended sites in the body. Organ specificity of delivery might be increased by the appropriate placing of external magnetic fields on catheters placed into the urethra or rectum to attract higher concentration of the systemic iron particles to the prostate or colon. Similar approaches could be adapted to treat other primary tumors in the breast or esophagus.

Cancer cell specific–delivery of these nanoparticles might involve molecular probes that attach the nanoparticles to tumor cell surface markers or to tumor endothelial cell markers. Prostate-specific membrane antigen is an attractive target for such direction to prostate cancer cells. Prostate-specific membrane antigen levels are markedly enhanced on the surface of advanced human prostate cancer cells that have failed androgen deprivation therapy. Tumor-specific antibodies and aptamers²⁴ have been developed to bind specifically to prostate-specific membrane antigen. Adding these binding agents to nanoparticles²⁵ or liposomes containing microscopic iron particles could be a major advance for specifically heating advanced human prostate cancer cells. This induced hyperthermia within the cancer cells should also enhance the cancer cells’ response to other forms of combination therapy, such as radiation, cytotoxic, and immunotherapy.

These new types of therapeutic approaches may one day extend the Lance Armstrong effect to the treatment of other types of advanced disseminated human solid tumors. Although this article focuses on the nuclear matrix as the prime target for temperature sensitivity of cancer cells, it is important to also evaluate other proposed models that have recently been reviewed for germ-cell chemosensitivity.^{26 - 27} To accomplish this goal, new concepts, better preclinical models, and precise clinical delivery and evaluation must be developed. These goals are within reach and these new concepts might provide a nidus for forming a much-needed multidisciplinary approach to develop this therapy.