Averting Chaos

By Elise Lamar, PhD

Searching for the evolutionary origins of cellular order provided Matt Gibson with unanticipated insights into tumor development.

In the animal kingdom, epithelia—layers of tightly packed cells—are ubiquitous. They stretch over every external and internal surface of animals’ bodies forming protective barriers; channeling liquids; secreting milk, enzymes, and hormones; and constructing structures as diverse as insect wings, fish gills and the human spinal cord.

Not surprisingly, most human tumors originate in epithelia. Collectively known as carcinomas, they emerge when something goes terribly wrong and the highly organized structure of epithelia starts to melt down. “When cells are confined in an epithelial layer, things stay nicely organized,” says Stowers Associate Investigator Matt Gibson, PhD, who uses simple model organisms to reveal the universal mechanisms animals rely on to construct tissues. But recently, he and his team discovered that if dividing cells fail to align properly within the orderly array of the existing epithelium, rogue cells can break free—bringing them a step closer to initiating cancer.

Gibson hadn’t set out to study tumorigenesis. Rather, the work—which bridges basic and translational science—originated in evolutionary and cell biological studies the Gibson lab began two years ago.

An unanticipated discovery

Epithelial cells are polarized; that is, their upper (apical) end differs from their bottom (basal) side. In a 2011 Current Biology paper, the Gibson lab’s Drosophila team used imaging to show that fly cells round up at the apical epithelial surface during cell division to allow the nucleus to move into that region of the cell.

Prior to those studies, Gibson had recruited postdocs to help pioneer a novel experimental system in his lab: the sea anemone, which builds tentacles (not wings) from embryonic epithelia. One member of that team was Aissam Ikmi, PhD, who was trained at the University of Paris-Sud in Orsay as a Drosophila geneticist, but had decided to tackle a new challenge.

From left to right: Matt Gibson, PhD, Liang Liang, and Yu-ichiro Nakajima, PhD

Pioneering sounds glamorous but, in fact, it means that one might spend tedious months creating a molecular tool kit from scratch, while colleagues working in established systems like Drosophila publish papers. Ikmi might have found himself in that position, but instead got an unforeseen reward.

While testing reagents he made from scratch, he discovered that nuclei in dividing sea anemone epithelial cells moved into the apical end, which ballooned out just like Drosophila cells did. “I knew I would face challenges in establishing tools needed to study biological problems in an emerging model organism,” Ikmi says. “But this work brought important insight to the history of life by elucidating how ancient multicellular animals are constructed at the cellular and molecular levels.”

Emily Meyer, a Gibson lab technician who was co-first author with Ikmi on the study, did the Drosophila work. “We had planned experiments in flies to gather this data,” she says, explaining that sea anemone work was initially viewed as a side project. “But these important findings gave evolutionary direction to the story.”

Gibson agrees that side-by-side comparisons of insect and sea anemone epithelial cells led to a much broader
perspective on this biological problem. “Previously, the nuclear movement we describe here was thought to occur primarily in cells of the vertebrate neural tube,” he says. “But we showed that a fundamentally similar type of epithelial cell division occurs in organisms as distantly related as fruit flies and sea anemones.”

Discovering why

Biology 101 students know that if an organism more primitive than a fruit fly, like a sea anemone, does something resembling what human embryonic brain cells do, that something must be important, with a capital “I.” The Current Biology paper established that flaring out at one end before cell division was Important. The question remained why.
Earlier work provided a hint: The group had observed that a dividing cell’s mitotic spindle—the web-like machinery that separates chromosomes into daughter cells— invariably orients itself parallel to the apical surface of the cell layer, suggesting that apical rounding might play a key role in setting the stage for proper alignment of the mitotic spindle.

To determine what holds the spindle in the correct position, Gibson lab postdoc Yu-ichiro Nakajima, PhD, the first author of a 2013 Nature study, used high resolution imaging to look inside epithelial cells developing into a fly wing. He observed that the two ends, or poles, of the mitotic spindle always sat near the septate junctions, regions of close contact between neighboring cells. There, two proteins called Discs Large and Scribble were juxtaposed to the spindle.

“The spindle in mitotic cells seemed to know the right position and direction to orient,” says Nakajima. “That
suggested that Discs Large and Scribble might provide the cue to orient at this position.” Given the Gibson lab’s dual expertise in genetics and cellular imaging the next step was clear–namely, to genetically delete Scribble and Discs Large in Drosophila embryos and watch what happens.

Viewing “misorientation”

Conventional microscopy revealed that Nakajima was right. Deleting Scribble caused the mitotic spindle to flop over at a random angle, as did loss of Discs Large. The group then perturbed the spindle with other reagents and video-captured outcomes using a microscope custom-built by Sean McKinney, PhD, and Amanda Kroesen of the Stowers Microscopy Center. Using methods pioneered by Gibson lab graduate student Liang Liang, that microscope allowed the team to embed living fly tissue in a gel and then spin it around while filming what happened for more than an hour.

Analysis of the resulting videos revealed that during division, misoriented cells peel away, or delaminate, from the epithelium. By contrast, videos of normal tissue show epithelial cells dividing, jostling their neighbors, and then settling in an orderly block.

Gibson says that when potentially harmful cells “fall out of the epithelium” they are generally killed by apoptosis, a self-policing mechanism that eradicates damaged cells. Nakajima, in fact, had studied apoptosis in Drosophila as a PhD student at the University of Tokyo. At his suggestion, the group experimentally blocked apoptosis and observed what happened in cells once spindle orientation was disrupted.

That manipulation produced the paper’s critical result: Misoriented cells that got an experimental reprieve from protective cell death produced tumor-like growths. Even worse, cells in those masses lost all semblance of normal polarity, or shape, and switched on fruit fly homologues of human carcinoma markers.

The answer to basic questions

As Gibson and Nakajima observed, the good news for flies, sea anemones, and humans is that tissues usually succeed in killing off cellular miscreants via apoptosis. But their lab’s work suggests that if apoptosis were short-circuited by mutations in genes required for good cell death—mutations common to almost all human cancers—cells ejected from an epithelium could switch on cancer genes and flee to a different tissue. The clinical term for that phenomenon is metastasis.

“If you disrupt mitotic spindle orientation, abnormal cells should typically die because epithelia have an intrinsic mechanism to protect themselves,” says Gibson. “But if this mechanism is compromised in a given individual, they would be vulnerable to potentially tumorigenic events.”

Overall, these studies prove that answering the most basic questions—such as what cellular mechanisms are conserved across millions of years of animal evolution—is the very basis of meeting translational goals of knowing what goes wrong in a cancer cell.