Stanford-developed molecule homes in on enzyme that promotes colon cancer tumor growth

Molecules created by Stanford Medicine researchers have revealed the secrets of cells thought to be involved in colon cancer relapse and resistance to chemotherapy. They may also be a springboard for new targeted cancer drugs.

As reported in Nature Chemical Biology on July 4, the molecules precisely target an enzyme called ALDH1B1, which is known to be overactive in patients with colon and pancreatic cancer. Prior studies in mice have demonstrated a role for ALDH1B1 in pancreatic tumor development by specifically acting in cancer stem cells. Treatments that target cancer stem cells may be especially critical for preventing cancer relapse and chemoresistance.

The general consensus on cancer stem cells is that these slow-growing cells can give rise to all other cell types in a tumor, said senior author James Chen, PhD, professor of chemical and systems biology, of developmental biology, and of chemistry.

“When tumors respond to chemotherapies that target the most rapidly dividing cells, the slowly dividing stem-like populations can persist and are the basis for tumor relapse and chemoresistance,” said Chen, the Jauch Professor.

If ALDH1B1 is required for cancer stem cell survival, then a drug that disrupts its function could be an effective therapy, he said. An advantage of such a drug would be precision: While chemotherapies kill any fast-dividing cells, including healthy cells, a targeted treatment that goes after a specific protein like ALDH1B1 can focus its attack on cancer cells. But to get there, scientists must first better understand ALDH1B1’s role in cancer.

“No one really understands why ALDH1B1 promotes colorectal and pancreatic tumor growth,” Chen said.

A new set of molecules

Unraveling the mystery of ALDH1B1’s role in cancer is now facilitated with the molecules developed by Chen and his team. Until now, there was no molecule known to specifically target ALDH1B1 — a member of a 19-enzyme family called aldehyde dehydrogenases, or ALDHs. In healthy cells, ALDH enzymes help clean up toxins called aldehydes that accumulate inside cells.

A few ALDH family members are known to be overproduced in cancer cells, and individual family members seem to have their own cancer niches, with one ALDH family member linked to ovarian cancer and another linked to colon cancer, for example. But the details of what ALDH enzymes are doing in cancer cells is murky.  

“It’s hard to study this kind of enzyme because their structures are very similar, and many of them share similar biochemical functions,” said lead author Zhiping Feng, a postdoctoral fellow on Chen’s team, referring to the ALDH enzyme family. The new molecules developed by Feng, Chen and colleagues are the first of their kind for targeting ALDH1B1 while leaving other ALDH family members alone.

This biochemical breakthrough may have been missed if not for a chance discovery. Originally, Chen’s team was looking for a molecule that could block another pathway that promotes cancer growth. After testing more than 300,000 synthetic compounds, the team thought they had a good candidate for their original goal. Unexpectedly, they discovered their candidate molecule interacted with a member of the ALDH family that is not involved in cancer. Given the structural and biochemical similarities of ALDH family, the team wondered if the candidate molecule and others like it could block the activity of other ALDH enzymes that are involved in cancer.

“To our surprise and satisfaction, one of our molecules preferentially targeted ALDH1B1,” Chen said. Although ALDH1B1 was not related to the pathway the team was originally pursuing, they continued to investigate the enzyme and the molecules that blocked its function.

The team next sought to optimize these molecules, improving their ability to suppress ALDH1B1 function while reducing side effects in cells. “It took almost two years to get to the final compounds,” Feng said. “We tested lots of different compounds, and some didn’t work at all.”

The team solved the first X-ray crystal structure of ALDH1B1, which enabled them to examine the atomic-level interactions between the candidate molecule and the enzyme. They then used these insights to guide chemical modification of the molecules, eventually testing over 100 different versions for their ability to block ALDH1B1 activity. During the course of these studies, the team also had to change the molecular backbone of the candidate molecules. The backbone of the original molecules caused toxic side effects in mitochondria, the cellular location where ALDH1B1 operates. The researchers found that a similarly shaped, alternative structure avoided this toxicity. The team named their hard-won candidate molecules after their chemical structure and precision attack on ALDH1B1: isoform-selective guanidinyl antagonists of aldehyde dehydrogenases, or IGUANAs.

ALDH1B1 and cancer

To understand whether colon cancer stem cells need ALDH1B1 to survive, the team used the IGUANAs in a battery of cell-based tests. One test showed that IGUANAs stopped ALDH1B1 activity in colon cancer cells grown in the lab. In another test, the team studied ALDH1B1’s function in stem-cell-like colon cancer cells.

For this test, colon cancer cells were grown in a traditional single layer as well as in three-dimensional balls called spheroids, which require stem-cell-like cells for their survival. When IGUANAs were added to block ALDH1B1 activity, the stem-cell-enriched spheroid cultures died, while the single-layer-cell culture was largely unaffected.

“This confirmed the suspicion that [ALDH1B1] is involved in stem cells,” Feng said, “and suggests ALDH1B1 is important for supporting cancer stem cell survival or maintenance.”

Finally, to explore how ALDH1B1 promotes colon cancer stem cell survival, the team looked at how other genes in colon cancer cells are affected when ALDH1B1 is blocked by an IGUANA or genetic methods. The absence of active ALDH1B1 diminished the activity of genes involved in mitochondrial energy production and the function of critical cellular structures called ribosomes. “These findings reinforce a growing appreciation that mitochondrial metabolism and certain ribosomal functions favor a stem-like state in cancer cells,” Chen said.

The IGUANAs could lead to a targeted cancer drug that acts independently or in combination with a chemotherapy, Chen said. In the study, the team showed that IGUANAs stymied the growth of colon cancer cells from patients but had little effect on non-cancerous cells. In another experiment, the team used a cocktail of an IGUANA and fluorouracil — a mainstay chemotherapy for colon cancer — and found the combination was more effective at killing colon cancer spheroid cells than fluorouracil alone. The IGUANAs even prevented fluorouracil-resistant cancer cells from forming stem-cell-rich spheroids.

The research team’s next goal, Chen said, is to further understand the role of ALDH1B1 in cancer cell stem cells and to obtain ALDH1B1-blocking molecules that are suitable leads for drug development. The latter efforts are now being supported by the Stanford Innovative Medicines Accelerator. His team would also like to create molecules capable of disrupting ALDH family members that are involved in other cancers.

“The hope is that the fundamental insights we gain through our research at Stanford will extend to new clinical therapies,” Chen said.

The work was supported by the Stanford SPARK Translational Research Program and the National Institutes of Health.