In Memoriam: Al Knudsen, a modest, under-recognized founder of cancer genetics (and more)

My first job was a young faculty member was in the Graduate School of Biomedical Sciences, at the University of Texas Health Science Center in Houston.  Our small Center for Demographic and Population Genetics was part of the Graduate School, and it was small enough that we got to know, and interact with, the Dean.  And what a dean he was!

The great, and good Al Knudsen (1922-2016).  Google images.

It was a small graduate school, so Dr Knudsen still was active in research, cancer research. One of the first talks I heard down there in Houston, when I still didn't have my first pair of cowboy boots, y'all, was an interesting idea about the causes of cancer.

Radiation was a known carcinogen, as were some chemicals, and there were various ideas about how carcinogenesis worked at the gene level. The basic idea was that these agents caused genetic mutations that led cells to misbehave, and though abnormal, escape detection by the immune system. More mutations meant more cancer risk, and this was consistent with 'multi-hit' ideas of cancer. More mutations took longer to accumulate, which was consistent with the increasing risk of cancer with age.  But genetics was still very rudimentary then, compared to now, direct testing primitive at best. And there were some curious exceptions.  An interesting fact was that some cancers seemed familial, arising in close relatives, and typically at earlier ages than the sporadic versions of what seemed to be the same type of tumor.  Why?

One example was the eye cancer retinoblastoma which arose in children or young adults, mostly in isolated cases; but there were affected families in which Rb was often present at birth.  Knudsen's idea was that in affected families one harmful allele was being transmitted, but the disease did not arise until a second mutation occurred.  Al published a quantitative mutational model of the onset age pattern in a PNAS paper in 1971, just before I myself had arrived in Houston, but by chance I had heard him present his work at the time of my job interview.

The basic idea was a 2-hit hypothesis, in which you could inherit one Rb mutation, and then only had to 'wait' for some one of your embryonic retinal cells to suffer the bad luck of a hit in the normal copy in order for a cancer to develop.  That waiting time accounted for the earlier onset of familial cases, because they only had to 'wait' for one mutation, whereas sporadic cases needed to experience two Rb hits in the same cell lineage.

This was a profound insight.  It allowed for cancer genetic findings, in which some forms of cancer clustered in families (e.g., some breast and colorectal cancers). Yet most cases were sporadic.  It was shown roughly at that time, by clever work in those crude days of human genetics, that tumors were clonal--the tumor, even when it had spread, was the descendant of a single aberrant (mutated) cell.

It did not take long for this sort of thinking, along with various methods for detection, to find the Rb gene....and other genes related to cancer.  This eventually included genomewide tests for loss of detectable variation based on microsatellite sites, continued to confirm the idea, far beyond those types of cancer that seem to be caused largely by changes in a single gene. The idea of somatic mutation caused by environmental factors, was complemented by the idea that it is common to inherit genotypes that are partially altered but insufficient by themselves to cause cancer, so that the tumor only arises later in life, after environmentally-caused (or stochastic) further mutations occur.

Knudsen's basically 2-hit idea was quickly generalized to 'multi-hit' models of cancer, and the discovery that cancers in a given individual were clonal led to models in which combinations of inherited mutations (present in every cell) and those that occurred somatically, seemed to account for the basic biology of cancer.  Many of the individual genes whose mutation puts a person at very elevated risk of one or more forms of cancer have since been identified, and newer technology has allowed their functional nature (and reason for their role in cancer) to be found.  Some are involved in DNA repair or control of cell division, and it's understandable why their mutational loss is dangerous.

The sources of variation in these genes may vary, but cancer as a combination of inherited and somatically generated mutations is a, if not the, prevailing general model for its biological nature and epidemiology, and shows why tumors are somatic evolutionary phenomena at the gene level.  But his nugget of an idea triggered much broader work in human genetics that, once technology caught up to the challenge, led to our understanding (and, too often, convenient ignoring) of the role of combined inherited and somatically induced variation as a major cause of the common, complex disorders for which genomewide mapping has become a routine approach.

I was still in Houston when Dr Knudsen moved to the Fox Chase Cancer Center in Philadelphia.  We missed him, but over the following decades he continued to contribute to the understanding of cancer.  His inspiring, gentle, and generous nature was an exception in the snake-pit that has become so common in the 'business model' of so many biomedical research circles.

Al's foundational work earned him many honors.  But he didn't get one that I think he richly deserved: his quiet, transformative role in understanding cancer, and the much broader impact on human genetics that followed as a result, deserved a Nobel Prize.

Do we still not know what causes cancer? Part V. Simple complexity?

The insightful somatic mutational idea of Al Knudson's that retinoblastoma (RB) was due to two mutational hits, that accounted for the onset at or near birth, was based on the single vs multiple, unilateral vs bilateral nature of the primary tumors, and the difference between sporadic and familial cases.  Knudson's two-hit model originally (way back in 1971) was basically about two different genes needing to be mutated.  At the time we knew little about cell-to-cell signaling environments and the like, nor about the kinds of genes--related to basic cellular function--that might be responsible.  As it turned out, the RB story seemed to be one of two mutations--but in the different copies of the same gene, that when discovered was named RB1.  It turns out to be a tumor suppressor or 'anti-oncogene', one of the general classes of cancer-related mechanisms that research has identified.

As we said in the previous posts in this series, a consistent picture of the generic nature of genetic factors, based on cells' context-related behavior, seemed to have emerged from this insight.  It turned out that RB and perhaps a few other childhood tumors fit this simple one gene model.  There were some problems, in that mice engineered to be RB1-negative did not consistently have RB nor sometimes any serious abnormality.  And RB1 is expressed in many different cells, but had little direct relevance to other cancers (tumors didn't appear in childhood in other tissues).  Even so, again for whatever reason, this seemed to be a single-gene, single-tissue problem.  The idea could be that cancer is causally simple, and the job was just to identify the genes in each case (some of us were writing, even then, that the age pattern of other cancers suggested that they seemed to require 'hits' in multiple genes).

Cancer research following up on this and many other leads made great strides in many areas of basic cell biology and cellular communication--because cellular communication or cells sensing and responding to their environment is what complex organisms are all about. Cell-specific gene expression, signaling, and other aspects of cellular behavior were reflected in the abnormal cellular behavior of cancer.  RB1 inhibits cells from dividing until they are 'ready', meaning until other aspects of their behavior and responses have been set up (see Wikipedia: Retinoblastoma protein).

New light on an old story
But now even the RB story is turning out to be more subtle, and perhaps more complex than we had thought.  A new paper and commentary in Nature reports the story.  The question was how do RB1-negative cells end up being cancer cells?  Why does the absence of this gene matter?

The authors of the paper searched for other mutations in the genome in RB1 negative RB tumor cells, looking for evidence of a second 'hit'.  They did not find accumulating mutations.  In fact, they found that the retinoblastoma genome is more stable than that most other human cancers sequenced to date.  Rather than mutational or structural variations, they found that epigenetic changes were common.  These involve chemical modification of the nucleotides in the cells, rather than DNA sequence changes, the usual definition of mutation.  The changes include methylation of DNA that affects nearby gene expression and modification of the proteins that wrap up DNA in the nucleus and in turn are used to unwrap the DNA in areas where genes need to be expressed.

These modifications are driven by specific mechanisms that patrol DNA and, in ways not yet very well understood, modify it in chosen places.  But they do this systematically across the genome, making in a sense wholesale changes at a single go.  Those changes are somehow tissue context specific.  But the process does not require each site to be modified having to wait for a very rare mutational event. 

How RB1 protein affects cell cycle changes is not fully understood, but one thing that epigenetic changes can do is operate much faster than 'waiting' for a series of damaging DNA mutations to occur.  This works because epigenetic changes must occur all over the genome, since specific tissues use (or don't use) many different genes scattered across the chromosomes.  And tissue behavior can change, requiring different expression patterns: so the expression-regulating epigenetic changes have to be fast, modifiable, and reversible.  Which we know that they are.  Thus this is a plausible mechanism for the early onset of RB.  The authors identified a gene called SYK that may be particularly involved.

By contrast, for example, BRCA1 mutations damage DNA repair mechanisms, so that the person inheriting or incurring mutations in that gene becomes vulnerable to other mutations in her cells,  unfortunately including cancer-related mutations, that the cell can't detect and fix.  This shortens the average 'waiting' time for enough changes that a badly-responding breast cell divides out of normal contextual control.  BRCA1 related breast cancer is usually earlier than other cases.

Many changes all at once?
This may account for the early onset of the tumor when clearly just one aberrant gene doesn't alter so many aspects of the control of cell division and response.  Instead, the missing RB1 protein allows other processes involving many other genes to become deregulated--without having to be mutated themselves.  It would mean that RB as a disease is, like other cancers, the result of many changes in cell behavior, not just one.  In a sense that is reassuring, that we are correctly understanding that the mechanism of proper cooperation (to use MT's favorite term!) is required for appropriate cell behavior--appropriate in the sense that the pattern of cooperation evolved over time to produce the differentiated organ systems that we have in our bodies.

What does this say about Knudson's original two-hit model?  It was both right and wrong.  RB is a simple one-gene disease from the mutational view, but it is not a carcinogenic process that involves only one gene.   RB is a complex tumor, like other tumors, involving the actions of many genes, and their interactions with their contextual surroundings.  It is genetic in the sense that it involves aberrant use of many  genes, but it is contextual in that the genes are normal genes but mis-expressed in their context.  In truth, this is how all cancers are.  It just happens that this is driven by a single gene--or, of course, it could be that this works only in families transmitting otherwise unknown variation in genes that are vulnerable to these effects of the RB1 protein--that we don't yet know and would, like any GWAS problem, be challenging to discover.

A dangerous mix
Overall, what we see is a mix of somatic or inherited mutation, that unfortunately modifies a cell in nonmutational ways, that are genetic in the functional sense, yet are context-related because they  make the cell behave is if its context were different from the normal retinal context.  This is a mix of the two basic ideas of causation (called SMT and TOFT) that the BioEssays debate was about, and that originally triggered this series of commentaries.  In earlier installments in this series, we largely took sides, against the TOFT view, because it seemed to be in denial about the role that huge amounts of evidence provide for somatic mutations.  But as we've said in other parts of the series, no one can seriously argue that cancer is only genetic in the mutational sense, nor only involving somatic mutations.  The combination of causes is what is important and, we think, the age of onset pattern shows this.

But one of the reasons the genetic theory (including both somatic and inherited mutations) is so strongly supported by the evidence is that a great deal of evidence shows that tumors are, by and large, clones:  no matter how the tumor has spread in the body, all its millions of cells are descendants of a single 'transformed' cell.  There is evolution and genetic change among these cells, but they would share one or more changes that were there when that cell was transformed.  This is, we think, the typical finding.  There is, in fact, a new paper about the clonal nature of cancer in Nature.

By contrast, in any local cellular environment there are typically millions of cells, so that a screwy environment would misdirect responses in a great many cells, and the result would be a large number of primary tumors, and they would all look genetically normal. And they would have no reason to look so genetically abnormal as cancer cells.  Of course, abnormal cancer cells induce other perfectly normal cells, such as vessel-building cells, to provide nutrients, oxygen, and so on to the tumor cells.  So the tumor is much more than just the transformed cells.  The Nature review makes this clear, and that context is part of the story, which is related in a way to the TOFT theory, even if the article is from a cellular-genetic point of view..

Yes, we do know what causes cancer--generically.  It's about basic biology and the complex mix of causation, cooperation, and context that is the nature of life.  It's conceptually simple, but complex, like most of life, in the details of each instance.