The surprising results of a study headed by scientists at the University of Southern California offer up new insights about how cancer cells may metastasize, and also suggest new therapeutic approaches for halting their spread.
The NIH-supported work centers on a cellular chaperone protein called GRP78—also known as BiP—which helps to regulate the folding of other proteins. Building on previous work by the team, which is headed by Amy S. Lee, PhD, professor of biochemistry and molecular medicine at the Keck School of Medicine of USC. The researchers used techniques including confocal microscopy and gene mRNA knockdown, to show that GRP78, which normally resides in the endoplasmic reticulum (ER) of cells, can also be shuttled into the nucleus in times of cell stress, where it regulates gene expression and pathways, ultimately allowing cancer cells to become more aggressive.
“Seeing GRP78 in the nucleus controlling gene expression is a total surprise,” said Lee, the study’s senior author and the Judy and Larry Freeman Chair in Basic Science research at the USC Norris Comprehensive Cancer Center. “When it comes to the basic mechanisms of cancer cells, this is something novel that, to my knowledge, no one has observed before.” The findings could represent a paradigm shift for cell biology, and have implications for cancer therapeutics research. Lee and colleagues published their findings in the Proceedings of the National Academy of Sciences (PNAS), in a paper titled “ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator,” in which they concluded, “Our study reveals a mechanism for cancer cells to respond to ER stress via transcriptional regulation mediated by nuclear GRP78 to adopt an invasive phenotype.”
Molecular chaperones are increasingly recognized as major regulators of cellular homeostasis in health and disease, the authors noted. The chaperone GRP78, encoded by the HSPA5 gene, is a member of the heat shock protein 70 (HSP70) protein family. Unlike other, cytosolic HSP members, GRP78 contains a signal sequence that targets it to the endoplasmic reticulum. As a major ER chaperone GRP78 plays a key role in folding and processing membrane-bound or secretory proteins, and previous studies by Lee’s team had also shown that when cells are under stress—due to COVID-19 or cancer—GRP78 gets hijacked, allowing viral invaders to replicate, and cancers to grow and resist treatment.
Through their new study, the USC researchers made the unexpected discovery that when cells are stressed, GRP78 which is “commonly overexpressed in cancer cells” migrates to the cell’s nucleus, where it alters gene activities and changes the behavior of the cell, allowing cancer cells to become more mobile and invasive.
The new discovery started as an incidental one. Ze Liu, PhD, a postdoctoral researcher in Lee’s lab and the study’s first author, was analyzing how GRP78 regulates a gene known as EGFR, which has long been linked to cancer. Liu noticed something surprising: GRP78 controls the gene activity of EGFR, raising the intriguing possibility that, while GRP78 was long thought to exist primarily in the ER of cells, the chaperone protein may have entered the nucleus and assumed a new role.
To confirm their hypothesis, Liu, Lee, and colleagues used confocal microscopy, which offers high-resolution 2D and 3D imaging, coupled with an advanced technique for capturing images of live cells, to directly observe GRP78 in the nucleus of lung cancer cells, as well as normal cells under stress.
The researchers also set out to learn more about what happens in a cell after GRP78 enters the nucleus. Using a sophisticated form of RNA sequencing they compared lung cancer cells engineered to over-express GRP78 in the nucleus, with cells lacking GRP78 in the nucleus, to help find out which genes were affected. “To our big surprise, we found that the key genes being regulated by GRP78 in the nucleus are mainly involved with cell migration and invasion,” Lee said. The authors also noted, “This study uncovers a molecular mechanism by which cancer cells respond to stress through nuclear translocation of GRP78/BiP, which assumes a role as a transcriptional regulator, allowing cells to adopt an invasive phenotype and impacting other pathways.” They also pointed out that GRO78 is upregulated in a wide range of cancers and is associated with aggressive growth, invasive properties, and therapeutic resistance.
They used several techniques, including biochemical analyses and mRNA knock-down of GRP78, which allowed them to identify the signal within GRP78 that enables it to enter the nucleus, and confirm that when GRP78 is present in the nucleus, it stimulates EGFR gene activity. EGFR is well known to play diverse roles in tumorigenesis, proliferation, survival, and metastasis in many different types of cancer, including lung cancer. “… we found that GRP78 knockdown consistently suppresses EGFR protein expression level in a wide variety of human lung cancer cell lines harboring different EGFR mutational and amplification status,” they wrote. And when they looked at a database for gene expression in more than 200 different human lung cancer cell lines, “we detected a statistically significant positive correlation between GRP78 and EGFR transcript levels.”
Their results showed that GRP78 binds to ID2, another cellular protein that typically suppresses genes (including EGFR), many of which allow cells to migrate. But when bound to GRP78, ID2 could no longer do its job. “… nuclear GRP78 can regulate expression of genes and pathways, notably those important for cell migration and invasion, by interacting with and inhibiting the activity of the transcriptional repressor ID2,” the team wrote. And without activity of the ID2 transcriptional repressor, cancer cells become more invasive. Interesting, the investigators noted, “Recently, it was reported that ID2 exerts tumor suppressor properties in lung cancer through its effects on cancer cell invasion and migration.”
The new results point to several potential approaches for cancer treatment, including downregulating the activity of GPR78 to suppress EGFR in lung cancer, or preventing GRP78 from binding to ID2. Their findings, they also commented, suggest that GRP78/BiP inhibitors may offer a therapeutic approach to suppress EGFR in various human lung cancer cells without the limitations of targeting specific mutations.
While the present study analyzed lung cancer cells, GRP78 plays a similar role in various types of cancers including pancreatic, breast, and colon cancer. So, it’s also possible that GRP78 may bind to other proteins in the nucleus that are critical for cancer, opening up a new line of research in cancer biology. “Additionally, there are likely other interacting partners with nuclear GRP78 beyond ID2 and other mechanisms that remain to be explored,” they noted.
The discovery that GRP78 can travel to the nucleus and assume new functions could have broad implications across the field of cell biology. Lee said it’s possible—even likely—that other proteins that typically reside in one part of the cell could, under stress or other triggers, migrate to another part of the cell and alter cell behavior in multiple ways. “This is a new concept,” she said. “The protein itself is the soldier that does the job, but now we’re thinking it’s not just about the soldier, but also where the soldier is deployed.”
Lee and her team are also studying drugs that can inhibit the expression or activity of GRP78. An ongoing study of theirs suggests that small molecules that inhibit GRP78, such as YUM70, may even be able to block GRP78 activity in the nucleus of cells.