On the HMS Quadrangle: Tackling basic science questions about cancer
Ask for directions to the Harvard Center for Cancer Biology, and you may find yourself wandering the Quadrangle of Harvard Medical School that houses the seven basic science departments. But you’re in the right place. The center is a cross-departmental organization of researchers who are tackling fundamental questions about the biology of cancer.
“Basic science discoveries in epigenetics, protein homeostasis, or other emerging fields of research could lead to new approaches to cancer treatment,” says Peter Howley, MD, chair of the Department of Pathology, whose laboratory studies the human papillomaviruses associated with cervical cancer. Of the many other outstanding cancer researchers on the Quad, the work of a few is featured here.
Recapitulating development and breast cancer in three dimensions
In the laboratory of Joan Brugge, PhD, chair of the Department of Cell Biology, three-dimensional culture systems help researchers see a richer, more lifelike representation of the organization of breast tissue. “In 3D, you can visualize a hollow lumen, for example, which you would never be able to see in a two-dimensional sheet of cells,” says Brugge. “The more closely you can mimic the organization and behavior of normal cells in the breast,” she explains, “the better model you have to examine the consequences of oncogenic insults that perturb or disrupt that natural process.”
During development, timely cell death through apoptosis gradually carves out a hollow lumen in the glands of the breast, creating a space ringed with epithelial cells anchored to the extracellular matrix (ECM). Using their 3D culture system, researchers identified the protein BIM as the trigger of this cell death. To validate their findings, they knocked out the BIM gene in the mammary glands of mice, which prevented or delayed the clearance of cells in the lumen.
This same cell death program is normally activated when epithelial cells stray from the ECM into the lumen. But this safeguard fails in cancer. To better understand tumorigenesis in the breast, researchers introduced the oncogenes HER2, cyclin D, and PI3 kinase, one at a time, into the 3D model and observed the effects.
During these experiments, researchers learned that for cells to proliferate outside their normal niches, they need one signal to drive proliferation, another to turn off the apoptotic death process, and a third to prevent repairs of the metabolic defects arising from matrix detachment. Moreover, they found that the oncogenes allowed hyperproliferation into the lumen by using the Erk-MAPK pathway to shut off BIM.
Brugge and her colleagues then examined human breast tumors obtained from pathologist Stuart Schnitt, MD (BIDMC) and saw that BIM was turned off in about 50 percent of the samples. With his guidance, researchers are now studying whether blocking BIM will restore the death program for the wayward, matrix-detached cells. “Our interactions with Stuart Schnitt have been really important in shaping our ideas and addressing whether the studies we do in culture models are relevant to cancer,” says Brugge.
Her lab recently received an Erk inhibitor – actually, an inhibitor of MEK kinase, directly upstream of Erk – so that researchers can test it in mouse models to see whether reversing the suppression of BIM will lead to tumor cell death.
In search of epigenetic enzymes as new drug targets
Like a grand maestro, epigenetics orchestrates which genes are to be expressed and which are to be silent at any given moment. These post-translational chemical modifications of DNA and histone proteins – the scaffold around which DNA is wrapped – actively control the expression of genetic information. While epigenetic modifications do not alter DNA sequence, explains Yang Shi, PhD, professor of pathology at HMS, they do survive mitosis and play a role in development as well as in many areas of pathology, including cancer. The silencing of a tumor suppressor gene like p16, for example, can sometimes involve epigenetic changes, he says. “The gene itself is fine, but becomes silenced because of a histone or DNA modification.”
Epigenetics also helps to explain some genetic mysteries, such as the differences between identical twins. Although they have matching genetic information, identical twins can have slightly different histone or DNA modifications, says Shi. “Over time these subtle differences develop into larger ones in response to the environment, exposing one twin to a disease but not the other.”
The primary interest of the Shi laboratory is discovering enzymes involved in epigenetic regulation and finding out which ones might be attractive drug targets in cancer or other diseases. Researchers focus on a specific class of enzymes called histone demethylases, which detach methyl groups from histone proteins. For decades, however, scientists believed that histone methylation was irreversible and, therefore, that demethylases did not exist.
That belief tumbled in 2004, when the Shi laboratory discovered the first demethylase, LSD1, and further studies linked this enzyme to gene repression. More recently, Shi and other researchers discovered the demethylase PLU-1. His lab is now conducting molecular and biochemical studies in cell cultures and human cell lines to understand the mechanism by which PLU-1 regulates proliferation and whether the enzyme is a causal factor in cancer, says Shi.
Genetic tools also help. His lab was one of the first to develop a DNA vector-based RNAi approach for silencing genes – a technology researchers use to examine the function of a gene and to determine whether it is necessary for cell proliferation, says Shi. “We want to know if bringing down the expression level of PLU-1 specifically affects cancer cells but not normal cells.” If so, PLU-1 – or other demethylases yet to be discovered – may prove viable targets for drug development.
Defects in cell cycle checkpoints linked to cancer
Checkpoint proteins coordinate the timing of events at each stage of the cell cycle to control proliferation, DNA replication, and chromosome segregation. Yet these same checkpoints are often the sites of mutations, says Wade Harper, PhD, Bert and Natalie Vallee Professor of Molecular Pathology. His laboratory studies cell cycle control and the key molecular players – ubiquitin ligases and deubiquitinating enzymes – that coordinate protein turnover via the ubiquitin-proteasome pathway.
Ubiquitin ligases attach the tiny protein ubiquitin to other proteins, thus marking them for modification – usually, degradation by proteasomes. In a counter move, deubiquitinating enzymes, or DUBs, detach ubiquitin from proteins. “The opposing activities of ubiquitin ligases and deubiquitinating enzymes determine the timing of a particular turnover event,” Harper explains. And timing is everything.
Ubiquitin-specific protease 44 (USP44), for example, is a critical regulator of the spindle checkpoint, which controls chromosome segregation to prevent aneuploidy. By removing ubiquitin from a key regulatory molecule, USP44 ensures that chromosomes are not separated until properly aligned on the mitotic spindle. Otherwise, a daughter cell receives one or more extra copies of a chromosome, which can contribute to birth defects and tumorigenesis.
Harper and long-term collaborator Stephen Elledge, PhD (BWH), the Gregor Mendel Professor of Genetics, discovered USP44, a new DUB, by screening a library of short hairpin RNAs targeting ~900 genes in the ubiquitin-proteasome pathway for those that control the spindle checkpoint (see Nature article). These libraries, developed by Elledge, Harper, and others, provide a powerful genetic tool for identifying new components in the very complex process of cell division and cell cycle checkpoints.
Harper explains that of ~100 DUBs in the human genome, many appear to antagonize the E3 ubiquitin ligases, in particular. Thus in related research, he is studying a class of these E3s known as the SCF family, which he and the Elledge lab discovered about a decade ago. One of its components, Fbw7, which is mutated in breast, ovarian, and endometrial cancers, controls the degradation of transcription factors – including c-Myc, c-Jun, and Notch proteins – and, therefore, the abundance of these oncogenes. “Normally, the SCF-Fbw7 complex rapidly turns over these oncogenic transcription factors in cells,” says Harper, “but a mutation in Fbw7 builds up higher levels and promotes oncogenesis.” Exactly how this happens is the subject of ongoing research in his laboratory.
Harper is quick to underscore the collaborations that drove the success of these projects and will accelerate discovery in the future. “One of the keys to advancing cancer research,” he says, “is bringing together scientists with different expertise – in large-scale genomics, functional biology, and biochemistry – to make it all happen.”
– Lonnie Christiansen