the the prepared mind”- Louis Pasteur
This is a story of how a lab problem led directly to a new biological discovery relevant to lung cancer, a disease that kills about 185,000 people each year in the United States. For many years, my lab has studied the molecular mechanisms that control the synthesis of an essential cell signal, bone morphogenetic protein 2 (BMP2). Precisely regulated BMP2 synthesis is indispensable for normal embryogenesis and adult physiology. Indeed, BMP2 is often synthesized at abnormal levels in adult pathologies, including cancers. Consequently, our overall objective is to understand the factors that control the level of BMP2, because these are putative drug targets for diseases that involve abnormal BMP2 synthesis.
These studies require comparable tissue culture cell lines that either do or do not synthesize BMP2. Several years ago, Shan Jiang, a recently graduated PhD student, began a survey of matched transformed and non-transformed cell lines. We chose these cell lines because BMP2 was induced in human tumors of the same type relative to adjacent normal cells. Shan contacted several local labs to obtain feeder stocks and set about measuring BMP2 RNA and protein in these cells. She also simultaneously tested these lines for a common tissue culture contaminant, Mycoplasma, the smallest known self-replicating bacteria.
We were not too surprised to find that many of the lines were contaminated. Others have estimated that 15% to 80% of cell cultures may be Mycoplasma-contaminated. We were astounded, however, to realize that the contaminated cells synthesized huge amounts of BMP2 RNA and protein relative to non-contaminated cells. Relative to previously studied chemical compounds — such as the vitamin A derivative retinoic acid — Mycoplasma was the best inducer of BMP2 expression that I had seen. By intentionally infecting cells, we then found that Mycoplasma induced BMP2 in cell types from many lineages, suggesting that Mycoplasma is a nearly universal BMP2 inducer.
Because BMP2 is a potent cell signal that can regulate cell differentiation, growth, and death, we had discovered one more good reason to be paranoid about cleanliness in the tissue culture room. Undetected Mycoplasma contamination would confound the interpretation of how BMP2 functions in any lab model of growth, differentiation and apoptosis.
However, our findings also had potential clinical ramifications. John Langenfeld, a colleague at UMDNJ-Robert Wood Johnson Medical School, had found that most non-small cell lung carcinomas (NSCLC) synthesize far more BMP2 than normal lung tissue or benign lung tumors. Experimentally increased BMP2 levels increase tumor growth of the A549 lung cancer cell line in nude mice, while anti-BMP2 antibodies or Noggin, a BMP2 antagonist, reduce both primary and metastatic tumor growth. BMP2 increases lung cancer cell line invasiveness, promotes signaling pathways known to stimulate proliferation (e.g., Erk 1/2; PI 3-kinase/mTOR), and increases blood vessel formation in tumor models. Not surprisingly, given these data, high expression of BMP2 mRNA in non-small cell lung carcinomas (NSCLC) has been found to be negatively associated with patient survival. Following John’s suggestion, we tested the effect of Mycoplasma on BMP2 expression in non-transformed, BEAS-2B immortalized bronchial epithelial lung cells.
Shan, a highly observant student, noticed that Mycoplasma-infected, but non-transformed, BEAS-2B began to look different from the clean cells. Instead of growing in nice, contact-inhibited monolayers, infected BEAS-2B cells changed morphology and piled up in clumps. Infected cells also grew significantly faster than the uninfected cells. This antisocial behavior typical of malignant, transformed cells was never observed in uninfected cells grown in parallel.
Because anchorage-independent growth in soft agar is a hallmark of oncogenic transformation, we examined the ability of Mycoplasma-infected cells to form colonies in soft agar. After 10 days, the infected BEAS-2B cells seeded in the plate formed large colonies. In contrast, the uninfected BEAS 2B cells never formed colonies. Loss of contact inhibition and the ability of the infected BEAS-2B cells to grow robustly when suspended in soft agar indicate that Mycoplasma infection transformed the BEAS-2B cells. We also treated the cells with a BMP2 inhibitor, both on plates and in soft agar, and showed that blocking the BMP2 signal reversed the growth promoting effects of Mycoplasma infection. These results showed for the first time that Mycoplasma transforms human lung cells and that BMP2 promotes the proliferation rate of Mycoplasma-transformed human lung cells.
John compared the abilities of non-infected and Mycoplasma-infected BEAS-2B cells to form tumors in vivo. Although BEAS-2B cells are immortalized, they are not transformed, and do not form tumors readily in vivo. John injected 10 million infected or non-infected cells subcutaneously into female NCJ athymic nude mice. Two weeks later, palpable tumors were observed exclusively in the mice injected with Mycoplasma-infected cells. In contrast, animals injected with the uninfected BEAS-2B cells were tumor free 14 weeks after injection. Histological analysis of the tumors indicated that the tumors were poorly differentiated, highly malignant, and invasive. These results demonstrated that Mycoplasma infection stimulated the progression of these human lung cells through both transformation and tumorigenicity.
Mycoplasma is associated with various human diseases including pneumonia, asthma, and cancer. At least 10 species can colonize the human respiratory tract and lungs. Improved detection methods have shown that healthy individuals are often colonized without obvious clinical effects. Because Mycoplasma are mutagenic and induce BMP2 and other cell signals, sub-clinical infections may exert a long-term, pro-cancer effect. In fact, Mycoplasma has been found in a variety of carcinomas including small cell lung cancer. We demonstrated for the first time that Mycoplasma infection also is highly transforming in human lung cells. Because antibiotic intervention is feasible, an association between Mycoplasma infection and even a subset of lung cancers could have a major impact on human health.
Lung cancer is an intractable disease. More people die from lung cancer than breast, colon, and prostate cancer combined. Despite advances in research, 85 percent of patients diagnosed with lung cancer will die from their disease. Our serendipitous finding that a common microbe induces BMP2 has revealed novel therapeutic approaches based on modulating BMP2 signaling and/or anti-Mycoplasma therapy.
Melissa Rogers earned her BS from Rensselaer Polytechnic Institute in Troy, NY, and her PhD from Brandeis University in Waltham, MA. She received postdoctoral training at the Dana Farber Cancer Institute and was a Hughes Fellow in the Department of Genetics, Harvard Medical School. She was assistant, then associate, professor in the Department of Biology at the University of South Florida (USF) in Tampa. In 2001, Dr. Rogers moved to her present position as associate professor of biochemistry and molecular biology at UMDNJ-New Jersey Medical School.
John Langenfeld earned his medical degree from Rush Medical School in Chicago. He completed a general surgery residency at UMDNJ, and thoracic surgery training at West Virginia University and Memorial Sloan Kettering Cancer Institute, followed by a research fellowship at UMDNJ and Memorial Sloan Kettering. He has been on the faculty of UMDNJ-Robert Wood Johnson Medical School since 1999, specializing in thoracic oncology surgery. His research focuses on lung cancer.