L-Plastin is One of 70 Signature Genes Used to Predict Prognosis of Breast Cancer Metastasis

[Guest post written by John Leavitt, Ph.D., retired Senior Scientist at LPISM in Palo Alto CA from 1981 to 1988; living in Woodstock, CT.  Leavitt has contributed several posts to the Pauling Blog in the past, all of which are collected here.]

John

John Leavitt

On August 24, 2016, the New York Times summarized the results of a Phase 3 clinical study of 6693 women with breast cancer. The outcome of this extensive clinical study was published in the New England Journal of Medicine on August 25, 2016. The clinical trial had been initiated ten years earlier on December 11, 2006 in Europe, (2005-002625-31) and on February 8, 2007 in the United States (NCT00433589). The study examined seventy select genes (seventy breast cancer “signature genes”) out of approximately 25,000 genes in the human genome that, when assayed *together* using a high density DNA microarray, predict the need for early chemotherapy.

In other words, the study asked which of the 6,693 tumors were “high risk” and likely to metastasize to distant sites within a five-year period, and which of these tumors were “low risk” and likely not to metastasize to distant sites in five years. One stated purpose of the study was to determine the need for chemotherapy, which can be very toxic and cause unnecessary harm to the patient, in treating breast cancer. The study found that a certain pattern of elevated or diminished expression of the seventy signature genes can predict a favorable non-metastatic outcome without chemotherapy for five years (while undergoing other forms of therapy such as surgery and irradiation).

One of the seventy selected genes is L-plastin (gene symbol “LCP1” and identified by the blue arrow in the figure below).

List of 70 signature genes

In 1985, my colleagues and I identified this protein in a cancer model system and named it “plastin” (Goldstein et al., 1985). We cloned the gene for human plastin while at the Linus Pauling Institute of Science and Medicine in 1987, and discovered that there were two distinct isoforms encoded by separate genes, L- and T-plastin (Lin et al, 1988). In 2014, in a piece published on the Pauling Blog, I described in some detail the discovery of L-plastin and its subsequent cloning.

A second figure, which is included below, summarizes information about L-plastin in a gene card published by the National Center for Biotechnology Information. This card shows that “LCP1: is the gene symbol for L-plastin and also identifies alternative names for L-plastin. Except for the inappropriate expression of L-plastin in tumor cells, this gene is only constitutively active in white blood cells (hematopoietic cells of the circulatory system). We used very sensitive techniques to try and detect L-plastin in non-blood cells such as fibroblasts, epithelial cells, melanocytes, and endothelial cells, but could not detect its presence in these normal non-hematopoietic cells of solid tissues.

Plastin Gene Card

The L-plastin gene card.

The clinical study reported on in the New York Times and New England Journal of Medicine shows that if L-plastin is not elevated in synthesis and modulated in combination with other signature genes, there should be little or no metastasis in five years. However, if L-plastin, in combination with other signature genes, is elevated in the early stage tumor, then the tumor is a high risk for metastasis and should be treated with chemotherapy.

plastin gels

The above figure consists of a pair of two-dimensional protein profiles that show the difference in expression of L-plastin and its phosphorylated form (upward arrows) between a human fibrosarcoma (left panel) and a normal human fibroblast (right panel).

My colleagues and I also found that L-plastin elevation is likewise a good marker for other female reproductive tumors like ovarian carcinoma, uterine lieomyosarcoma and choriocarcinoma (uterine/placental tumor), as well as fibrosarcomas, melanomas, and colon carcinomas. Abundant induction of L-plastin synthesis was likewise observed following in vitro neoplastic transformation of normal human fibroblasts by the oncogenic simian virus, SV40 (see Table IV in Lin et al, 1993).

The abundant synthesis of L-plastin that we found normally in white blood cells (lymphocytes, macrophages, neutrophils, etc.) suggested to me that the presence of L-plastin in epithelial tumor cells like breast cancer cells contributes to the spread of these tumor cells through the circulatory system to allow metastasis at distant sites. Indeed, both plastin isoforms have now been linked to the spread of tumors by metastasis, an understanding that is summarized in another Pauling Blog article from 2014 and, more recently, in other studies.

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The Discovery of Human Plastin at the Pauling Institute

Milestones in Plastin Research

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

In 1985 my lab at the Linus Pauling Institute of Science and Medicine (LPISM) in Palo Alto, California started to work on an abundant protein of white blood cells (lymphocytes, macrophages, etc.) that mysteriously appeared in human tumor-derived cells of solid tissues (carcinomas, fibrosarcomas, melanomas, etc). I had noticed this phenomenon a few years earlier while at the National Institutes of Health. I also noticed that this protein appeared in oncogenic virus-transformed (SV40 virus) human fibroblasts, but the protein was not expressed in the normal fibrolasts.

I was intrigued by the fact that a major protein of circulating blood cells would be induced during solid tumor cell development because it is well known that solid tumor cells become more anchorage-independent and can circulate like white blood cells to metastasize to other organs. My colleague, David Goldstein, took the lead in examining the expression of this mysterious protein in different cell types of fractionated white blood cells. At the time this protein was assigned only a number (p219/p220) corresponding to its position in two-dimensional protein profiles. We found that this protein was abundantly expressed in all normal white blood cell types that we examined but it was not expressed in normal cells of solid tissues (Goldstein et al, 1985).

When David’s paper was submitted to Cancer Research, the reviews came back positive and the paper was accepted for publication, but one reviewer asked that we give the protein a name. I was thrilled by the thought of naming a protein and its gene which would immortalize our work, so I took on the serious task of coming up with a name that had lasting meaning. My theory was that this cancer marker contributed in some then-unknown way to the plasticity of the cytoplasm in solid tumor cells because of its normal presence in circulating white blood cells. Also, I had seen the great movie, The Graduate, with Dustin Hoffman and recalled that amusing scene depicted in the picture included below. So I named the protein “plastin” – the greatest new thing since sliced bread. 🙂

The Graduate

That same year, I met Steve Kent from Caltech at a meeting in Heidelberg, Germany. After hearing my talk, Steve suggested that we collaborate. He mentioned that a postdoctoral fellow in Leroy Hood’s lab, Dr. Ruedi Aebersold, was trying to develop a more sensitive protein sequencing method for purposes of determining snippets of amino acid sequences from small amounts of unknown proteins eluted from two-dimensional gels (protein profiles) like the gels that we used to characterize plastin in David’s paper. If we could get an accurate partial sequence of plastin, we could devise a nucleic acid probe based on the genetic code that could be used to clone a plastin “copy DNA” from a cDNA library. If the plastin cDNA was cloned, we could then define the protein and perhaps its function by determining the nucleic acid coding sequence in the clone.

Madhu Varma.

Madhu Varma.

I gave Dr. Madhu Varma at LPISM the arduous task of isolating the plastin polypeptide “spot” for sequencing. Madhu cut out the stained spot from 140 two-dimensional gels, in effect purifying enough protein for sequencing by Ruedi at Caltech. Madhu succeeded and Ruedi produced eight short peptide sequences that could be used to develop short nucleic acid probes that would hybridize to the plastin cDNA clone isolated from a tumorigenic human fibroblast cDNA library.

Ching Lin.

Ching Lin.

Dr. Ching Lin at LPISM took one of the nucleic acid probes and immediately attempted to screen a tumorigenic fibroblast cDNA library. If we identified any clones that bound this probe, then we would perform a quick test to determine that we had cloned the plastin coding sequence. But science is full of surprises and we found that the first clone he isolated detected a gene product that was not in lymphocytes but only in normal human fibroblasts – in other words, it failed the test. This is where Ching’s brilliance took over. He was convinced that this first clone he had isolated was indeed a plastin coding sequence so he used this clonal DNA as a new probe against the tumorigenic fibroblast cDNA library. He isolated a new clone that passed the test and detected a gene that was expressed in lymphocytes and tumorigenic fibroblasts but not in normal human fibroblasts.

We performed other experiments that proved that we had cloned two different isoforms of plastin: L-plastin, expressed in lymphocytes and solid tumor-derived cells, and T-plastin that was expressed in normal solid tissues and co-expressed with L-plastin in tumor cells from solid tissues (Lin et al, 1988; Lin et al, 1990). Ultimately this work led to the complete characterization of the human plastin multigene family and verification that both isoforms were aberrantly expressed in various types of human tumors.

The figure at the top of this post maps the progression of discovery that followed our research, which began at the Pauling Institute in 1985. Our publications are shown in red in the graph and research published by other labs is shown in the blue bars.

Here are several plastin milestones discovered by other researchers:

  • T-plastin is abundantly induced in Sezary lymphomas, a lethal T-lymphocyte cancer (Su et al, 2003);
  • L-plastin induction in solid tumors contributes to invasive cancer growth and metastasis (Klemke et al, 2007);
  • Mutations in T-plastin play a role in the genetic disease Spinal Muscular Atrophy (Oprea et al, 2008); and
  • Most recently mutations in both L- and T-plastin promote re-growth of colon carcinomas following surgical resection of these tumors and chemotherapy (Ning et al, 2014).

These developments are more or less typical of the way science works. Progress in understanding complex phenomena like human cancer is the work of many scientists that builds on the observations of other scientists. This is just one example of the productive contributions in biomedical research that came about through early discovery research at LPISM in the 1980s.

Pioneering the Field of Proteomics

John Leavitt, 1982.

John Leavitt, 1982.

[Guest post written by John Leavitt, Ph.D., Nerac, Inc., Tolland, CT.]

In the fall of 1985, I went to a small meeting in Heidelberg, Germany, with Steve Burbeck from the Linus Pauling Institute of Science and Medicine, who had helped me by developing computerized microdensitometry to analyze two-dimensional protein profiles. At this meeting I described our protein profiling work and the discovery of the mutant beta-actins and another interesting protein which I named “plastin.”

Steve Kent, head of the protein sequencing facility in Leroy Hood’s lab at Caltech, heard my talk. We sat across from each other at dinner and he proposed a collaboration to develop methods of sequencing minute amounts of protein leached from spots in high resolution protein profiles. Lee Hood was well known for developing state-of-the-art protein and nucleic acid sequencing methods and machines, and was a founder of Applied Biosystems in Foster City, CA.

After I returned from Heidelberg, Ruedi Aebersold called me from Caltech and we began collaborating on microsequencing of pure nanomolar quantities of unknown proteins of interest eluted out of my protein profiles. In this work we essentially started the field of proteomics, which was eventually named ten years later by Jim Garrells, a protein profiler at Cold Spring Harbor. Proteomics is the search for and definition of proteins that could serve as diagnostic markers and drug targets for diagnosis and treatment of diseases, in our case cancer.

In 1987 we published a landmark paper in PNAS on the microsequencing technique that Ruedi developed. This paper would eventually be cited in references by more than 1,000 other research papers.

Ruedi

I gave a postdoctoral fellow, Mahdu Varma, the task of isolating the cancer-specific leukocyte isoform of plastin (L-plastin) from 140 protein profiles. This protein has been implicated in metastases in both melanoma and prostate cancer as well as in other aspects of cancer. The L-plastin spot was easily recognized and those spots on a nitrocellulose filter were “snipped out,” removing all the other proteins of the cell. We sent Ruedi a plastic tube containing the 140 “spots” of L-plastin. He had figured out a way to solubilize the protein from the nitrocellulose and was successful in determining the sequence of eight oligopeptides of between eight and sixteen amino acids derived by digestion of L-plastin with a proteolytic enzyme.

The peptide sequences he determined turned out to be perfectly accurate internal amino acid sequences of plastin when we decoded the sequence of the plastin gene (cDNA) clone, a reverse transcript of the messenger RNA. This was the first time that anyone had done this and it opened up the field of proteomics and led to the discovery of other diagnostic and drug targets.

plastin 1

We had chosen L-plastin, normally only expressed in white blood cells, because I had reported for years that it was a cancer marker in tumors that arose in solid tissues (identified in the image above by the two upward arrows). After we received the oligopeptide sequences from Ruedi, we made short DNA antisense probes that would hybridize to DNA sequences encoding these peptides in the human genome to fish out the full-length DNA clones that carried the sequence of the L- plastin gene.

Ching Lin and I, along with Reudi, published the sequences of the human L- and newly discovered T-plastin proteins, based upon sequencing of cDNAs, in Molecular and Cellular Biology. The discovery of a second isoform of plastin (T-plastin named for tissue plastin as opposed to L-plastin from leukocytes) was a surprise. We now had two genes to characterize at the genomic level. Today, T-plastin is a well recognized marker for cutaneous T-cell lymphoma (Sezary Lymphoma) and L-plastin, inappropriately expressed in solid tumor cells (carcinomas, fibrosarcomas, etc.), is understood to be a contributor to metastasis.

The Linus Pauling Institute was not all work and no play in the 1980s

We worked hard at the Institute and Linus Pauling was always there and visible.

We put together a softball team with Jim Fleming, Dan McQueeny, Zelek Herman, myself, and others at the Institute and played departmental teams at Stanford. I think we were called the “Pauling squeeze.” After these games we would often go dancing at the Class Reunion on El Camino Real near the corner of Page Mill Road.

We were fortunate to have on staff a first rate fundraiser in Richard (Rick) Hicks who arranged wonderful parties on Nob Hill at the Stanford Court. The most memorable of these parties occurred in late November 1986, when we honored Japanese billionaire Ryoichi Sasakawa with the annual Linus Pauling Medal. Another year Carl Sagan and Ann Druyan, who helped Carl put together the Cosmos series, likewise took part. We often saw Dr. Pauling’s sons, Linus Pauling Jr., Peter, and Crellin as well.

Here we are at the Stanford Court that night with postdoctoral fellows, Dr. Karin Sturm from Heidelberg, Germany, on the left and Dr. Madhu Varma from Madras, India, on the right. My wife, Becki, is in the middle. I recall that Dr. Pauling enjoyed this night as well.

Here we are at the Stanford Court that night with postdoctoral fellows, Dr. Karin Sturm from Heidelberg, Germany, on the left and Dr. Madhu Varma from Madras, India, on the right. My wife, Becki, is in the middle. I recall that Dr. Pauling enjoyed this night as well.

In 1988 I moved on to a new position in San Jose and then became Director of Research at Adeza Biomedical. Since we continued to live in Palo Alto, we continued to interact and party with the Linus Pauling Institute staff into the 1990s.