Frisch and Francis weren't searching for new cell death pathways when they chanced on anoikis. The pair was scrutinizing a bizarre adenovirus protein called E1a that can restore tumor cells to normal behavior. The researchers observed that tumor cells “reverse transformed” by E1a die when they separate from the ECM. E1a also bestows some epithelial characteristics on the reverse transformed cells, so Frisch and Francis decided to test whether disengagement from the ECM is fatal for normal epithelial cells.
They transferred normal epithelial cells to culture dishes coated with a compound that prevents cellular attachment. On gels, DNA from the free-floating cells showed a “ladder” pattern of equal-sized snippets, a telltale sign of the DNA degradation that occurs during apoptosis (Frisch and Francis, 1994). The researchers also detected breakdown products of the nuclear lamina, a sign that the nuclear envelope was disintegrating, another hallmark of apoptosis.
To confirm that separation from the ECM triggered death, the researchers raised cells on a congenial surface and then added small proteins that obstructed integrins, the membrane receptors that interact with the ECM. The cells died in droves. Frisch and Francis also showed that transforming cells with an oncogene or turning up production of the antiapoptotic protein bcl-2 staved off anoikis. The results “confirmed that epithelial cells are programmed to undergo apoptosis if they lose contact with the matrix,” says Frisch. The researchers chose a Greek term meaning “homelessness” for the phenomenon. Meredith et al. (1993) discovered a similar mechanism in isolated endothelial cells.
Eliminating wayward epithelial cells makes sense, Frisch explains, because they might grow in an inappropriate location. Still, they had to win over skeptical reviewers—it took more than a year to convince a journal to publish the report, he remembers.
Researchers have since learned that anoikis is ubiquitous. It helps cull excess cells in the digestive system (Hall et al., 1994), keeps the milk-producing bulbs in mammary tissue open (Debnath, et al., 2002), and helps hollow out the embryo early in development (Coucouvanis and Martin, 1995). Other scientists are piecing together how detachment causes death. For example, when cells break away from the ECM, they unshackle a protein called Bmf that detains survival-promoting molecules in the cell (Puthalakath et al., 2001). Frisch and colleagues showed that they could quell anoikis by inducing cells to produce a hyperactive version of the protein FAK, which flips on when cells attach to the ECM and promotes growth and survival (Frisch et al., 1996). This discovery was telling, Frisch says, because tumors often pump out extra FAK (Agochiya et al., 1999), suggesting that many cancer cells can't leave home without it. Other tumors ramp up production of TrkB, a protein that helps nurture nervous system cells, and recent work suggests TrkB allows intestinal cells to elude anoikis (Douma et al., 2004).