Cancer Explained – The Role of Cell Death

Following a recent blog, I received an inquiry from one of our readers. The individual asked whether I could better explain my oft repeated statement that “cancer doesn’t grow too much, it dies too little.” The questioner was puzzled by my assertion that chemotherapy drugs acted to stop cells from growing, while she had come to believe that this was synonymous with killing them. This dichotomy is at the crux of our modern understanding of cancer.

In response, I would like to examine the very basis of what is known as carcinogenesis, the process by which cancer comes to exist.

For more than a century, scientists believed that cancer cells were growing more rapidly than normal cells. They based this on serial measurements of patient’s tumors, which revealed that tumor dimensions increased. A small lump in the breast measuring one-half inch in diameter would be found six months later to be one inch in diameter. And six months after that it was two inches in diameter. This was growth, plain and simple, and so it was reasoned that cancer cells must be growing too much. As such, cancer therapies, per force of necessity, would need to stop cancer cells from growing if they were to work at all.

Dying Cell - lo resAnd then, in 1972, a paper was published in the British Journal of Cancer that described the phenomenon of apoptosis, a form of programmed cell death. Although it would be almost a decade before cancer researchers fully grasped the implications of this paper, it represented a sea change in our understanding of human tumor biology.

Let’s use the example of a simple mathematical equation. Every child would recognize the principles of the following formula:
Tumor mass = growth rate – death rate
This simple equation represents the principle of modern cancer biology. Where cancer researchers went wrong was that they mistakenly posited that the only way a tumor mass could increase was through an increase in the growth rate. However, as any child will tell you, a negative of a negative is a positive. That is, at a given growth rate, the tumor mass can also increase if you reduce the death rate. Thus, the “growth” so obvious to earlier investigators did not reflect an increase in proliferation but instead a decrease in cell attrition. Cancer didn’t grow too much it died too little, but the end result was exactly the same.

It should now be abundantly clear exactly why chemotherapy drugs, designed to stop cells from growing, didn’t work. Yes, the drugs stopped cells from growing, and yes any population of “growing cells” would suffer the effect. But they didn’t cure cancers because the cancers weren’t growing particularly fast. Indeed, the fact that chemotherapy works at all is almost an accident. Contrary to our long held belief that we were inhibiting cell proliferation, chemotherapy drugs designed to damage DNA and disrupt mitosis, were actually working (when they did at all) by forcing the cells to take inventory and decide whether they could continue to survive. If the injury were too extreme, the cells would commit suicide through the process of cell death. If the cells were not severely damaged or could repair the damage, then they carried on to fight another day. None of this, however, had anything to do with cell growth.

What is Cancer?

This is a question that has vexed scientific investigators for  centuries, and for the last century, our belief was predicated upon physical observation that cancer reflected altered  cell growth. After all, to the untrained eye, or even to the rather sophisticated eye, the mass in the pelvis or the lymph node under the arm, or the abnormality on a chest x-ray, continued to expand upon serial observation. This was “growth” (at least since the time of Rudolph Virchow); and growth it was reasoned represented cell division.

Based upon the cell growth model, cancer therapists devised drugs and treatments that would stanch cellular proliferation. If cells were growing, then cells needed to reproduce the genetic elements found in chromosomes leading to the duplication of the cell through mitosis. If chromosomes were made of DNA, then DNA would be the target of therapy. From radiation to cytotoxic chemotherapy, one mantra rang through the halls of academia, “Stop cancer cells from dividing and you stop cancer.”

As in many scientific disciplines, nothing spoils a lovely theory more than a little fact. And, the fact turned out to be that cancer does not grow too much, it dies too little. Cancer doesn’t “grow” its way into becoming a measurable tumor, it “accumulates” its way to that end.

In 1972, we realized that the most basic understanding of cancer biology up to that point was absolutely, positively wrong.

Working in a laboratory during my fellowships, I began to realize that something was wrong with the principles that guided cancer therapeutics. My first inkling came from the rather poor outcomes that many of my patients experienced despite high-dose, aggressive drug combinations.

Then, it was the failure of the clonogenic assay to predict clinical outcomes that further raised my suspicions. I began to ponder cell growth – cell death, cell growth – cell death. With each passing day the laboratory analysis that I conducted identified active treatments that worked.  Using short-term measures of cell death (not cell growth),. I could predict which of my patients would get better.  All of the complicated and inefficient clonogenic assay investigations could not. Cell growth – cell death – what was I missing?

It would be years before I would attend a special symposium on the topic of cell death that it all became abundantly clear.

My “eureka” moment is captured in Chapter 6 of my soon-to-be-released book, Outliving Cancer.FINAL book cover-lo res

Forms of Cell Death

Following the description of apoptosis in the British Journal of Cancer in 1972, scientists around the world incorporated the concept of programmed cell death into their cancer research. What is less understood is the fact that apoptosis is not synonymous with programmed cell death. Programmed cell death is a fundamental feature of multicellular organism biology. Mutated cells incapable of performing their normal functions self-destruct in service of the multicellular organism as a whole. While apoptosis represents an important mechanism of programmed cell death, it is only one of several cell death pathways. Apoptotic cell death occurs with certain mutational events, DNA damage, oxidative stress and withdrawal of some growth factors particularly within the immune system. Non-apoptotic programmed cell death includes: programmed necrosis, para-apoptosis, autophagic cell death, nutrient withdrawal, and subtypes associated with mis-folded protein response, and PARP mediated cell death. While apoptotic cell death follows a recognized cascade of caspase mediated enzymatic events, non-apoptotic cell death occurs in the absence of caspase activation.

With the recognition of programmed cell death as a principal factor in carcinogenesis and cancer response to therapy, there has been a growing belief that the measurement of apoptosis alone will provide the insights needed in cancer biology. This oversimplification underestimates the complexity of cell biology and suggests that cancer cells have but one mechanisms of response to injury. It has previously been shown that cancer cells that suffer lethal injury and initiate the process of apoptosis can be treated with caspase inhibitors to prevent caspase-mediated apoptosis. Of interest, these cells are not rescued from death. Instead, these cells committed to death, undergo a form of non-apoptotic programmed cell death more consistent with necrosis. Thus, commitment to death overrides mechanism of death.

Labs that focus on measurements of caspase activation can only measure apoptotic cell death. While apoptotic cell death is of importance in hematologic cancers and some solid tumors, it does not represent the mechanism of cell death in all tumors. This is why we measure all cell death events by characterizing metabolic viability at the level of cell membrane integrity, ATP content, or mitochondrial function. While caspase activation is of interest, comparably easy to measure and useful in many leukemias and lymphomas, it does not represent cancer cell death in all circumstances and can be an unreliable parameter in many solid tumors.

The Primacy of Microspheroids

After incorporating the realization that cancer biology was predicated on cell survival and not cell growth into our laboratory platform, we moved away from proliferative end points to cell death measures, and then redoubled our efforts to recreate the human tumor micro-environment in tissue culture. We immediately recognized that this required the preservation of cell-cell interactions found normally in the body as cellular clusters.

These cellular clusters better known as microspheroids, represent cohesive populations that interact directly with stroma, vasculature, inflammatory cells, and other tumor cells. Thus, the microspheroid recapitulates the human tumor environment. By applying cell death endpoints (the most rigorous of predictive measures) to these microspheroids, we have overcome most of the pitfalls encountered by earlier technologies. And, for the first time, a truly predictive human tumor model has been developed.

Of the two fundamental changes that we as a laboratory have brought to the field of chemosensitivity-resistance testing, the maintenance of cancerous tumor cells in their “native state” as microspheroids has been fundamental to our success.

Despite these important advances, many physicians have not grasped their significance. Falling back on their out-dated understanding of chemosensitivity studies that used growth-based endpoints (clonogenic, growth-to-confluence, and H3* thymidine incorporation, etc.) many physicians have failed to incorporate the use of these highly validated methodologies into their clinical practices.

A review of the published literature, correlating these more rigorous predictive methodologies with clinical outcomes, clearly establishes the validity of cell death in microspheroids as an important breakthrough in cancer treatment.

Chemosensitivity Testing That Makes Sense

Much of the controversy that has surrounded chemosensitivity-resistance assays (CSRA), reflects the fact that the majority of these tests were developed based on the erroneous belief that cancer was driven by its proliferative capacity and that the most active drugs could be chosen based upon their capacity to inhibit cancer cell growth. This led to a long series of unsuccessful attempts to predict clinical response based on cell proliferation endpoints.  Since the 1980’s we have come to realize that cancer represents a dysregulation of cell death and that effective drugs must kill cells outright (not inhibit their growth) in order to provide clinical response to patients.

The Ex Vivo Analysis of Programmed Cell Death (EVA-PCD) ® assay developed by Rational Therapeutics pioneered the application of drug induced cell death for the prediction of clinical response in cancer patients.  The EVA-PCD® assay was the first to incorporate this new understanding of cancer biology. By expanding the application of the EVA-PCD® platform to targeted therapies, RTI is now exploring new classes of compounds that function by inhibiting survival signals in cancer cells.  Many signaling pathways like the epidermal growth factor receptor (EGFr) have extracellular domains that function as cellular switches activating downstream phosphorylations following receptor ligation by proteins like EGF, amphiregulin and TGF alpha.

These mitogen activated protein kinases (MAPK) induce additional cascades of phosphorylations ultimately signaling transcription factors at the level of DNA. While these phenomena were originally thought to represent mitotic events, it is now recognized that most cells are not actively dividing, yet require all of these signaling pathway activations to remain alive. Thus, what was once described as growth factors are more likely better described as anti-death factors.

If indeed cancer doesn’t grow too much but dies too little, it is evident that effective therapies induce cell death, not growth inhibition in the patient.  This is why it is critical to apply lab analyses that measure cell death. Furthermore, as most of the signals for cell survival emanate from the extracellular environment, it is clear that cancer cells must be maintained in their native state to provide clinically relevant information. This is the basis of RTI’s human tumor microspheroid assay platform.