Summary of CMS Seminar Club presentation on Friday, November 25, 2022.
Title: Cell competition between normal and transformed epithelial cells
Speaker: Prof. Yasuyuki Fujita, MD & PhD, Kyoto University Graduate School of Medicine, Department of Molecular Oncology, Kyoto, Japan
On Friday, November 25, Prof. Fujita gave a presentation at Fujita Health University. He showed us how within an epithelial cell layer the healthy cells can get rid (induce apoptosis or apical extrusion) of cells with cancerous mutations (which are “transformed”). Then, the normal cells are the “winners,” and he believes this to be an important part of anti-cancer resistance. On the other hand, if cancer cells acquire more mutations, they can get the upper hand in this battle—become the winners—and kill or engulf neighboring cells and leave the epithelial layer from the basal membrane side so that they can spread to (metastasize) elsewhere in the body.
The story was beautifully and clearly explained, as Prof. Fujita is a natural educator. There were 24 participants who enjoyed the meeting. I only heard very positive reactions.
Recording: For members of Fujita University, a recording of the meeting (without the discussion part) will be available at our Manabi system. Unfortunately, we cannot open the recording for a wider audience.
For me, it was a joy to watch this recording again, because the experiments and their results were very clear while the implications are far-reaching. In this sense, it reminded me of the presentation last September by Dr. Oudhoff, who from another viewpoint also described that epithelial cells have their own ways of fighting (against pathogens in that case) apart from relying on immune cells. The original research papers by Prof. Fujita explaining the background of his presentation are also a joy to read because of their clarity and thoroughness.
The contents of the presentation
The below tries to summarize most but not all of Prof. Fujita’s presentation. In addition, I like to point out that Prof. Fujita has written excellent reviews on the topic (Kajita and Fujita, 2015; Kon and Fujita, 2021; Maruyama and Fujita, 2022).
The central question
What happens at the early stages of epithelial cells developing into cancerous cells, when some but not all mutations have been acquired that are necessary for becoming an aggressive tumor? How do those cells interact with neighboring epithelial cells?
Although some facts are known now, that is still a big puzzle.
History
Cell competition between epithelial cells in a monolayer was first described in Drosophila by Morata and Ripoll, 1975. That was a study on wing disc epithelial cells. Morata and Ripoll found that minute mutant epithelial cells underwent apoptosis if surrounded by wildtype epithelial cells but could survive normally if surrounded by minute mutant epithelial cells.
Prof. Fujita was the first to show that this kind of competition within epithelial layers also is used by normal epithelial cells to dispose of cells in their midst with cancerous (oncogenic) mutations (Hogan et al. 2009).
Currently, as Prof. Fujita summarized for us, this kind of eliminating competition within epithelial layers has been found based on various types of differences between cells. These possible differences can—for example—involve metabolism, senescence, infection, or species of origin. The summary in this blogpost, as Prof. Fujita did in his talk, will mostly focus on the competition between similar epithelial cells with and without a cancerous mutation. However, it is important to realize that for this type of competition to occur it does not specifically require differences in oncogenes.
Effect of single gene (-expression) changes in vitro
Mutations in Ras proteins that lead to uncontrolled cell growth are very common in tumors. As one of their model system, Prof. Yasuta and his group use Madin-Darby canine kidney (MDCK) epithelial cells that express GFP-labeled oncogenic Ras (RasV12) in a tetracycline-inducible system (Hogan et al. 2009). They found that if these cells are surrounded by normal MDCK cells in a monolayer on a collagen matrix, induction of RasV12 expression leads to extrusion of the living cells from the apical side of the monolayer (Fig. 1A), either as single cells or after forming clumps of cells. If RasV12-positive cells are grown together without being surrounded by normal cells, they do not show the extrusion behavior although their cell morphology is different and their cell-cell contacts are weaker than in a non-mutated monolayer.
Prof. Fujita showed a very nice movie about this apical extrusion (from Hogan et al. 2009), of which some screenshots are shown in Fig. 2. My computer settings have difficulties opening the movies from the Hogan et al. 2009 article, but there is a nice video from Prof. Fujita on-line where in 3 min he speaks (in Japanese) along this cell-culture movie (Academic crowdfunding platform “academist”).
The molecule Mahjong (aka VprBP or DCAF1) was discovered by the group of Prof. Fujita (Tamori et al. 2010) and named after a very competitive Chinese table game that Prof. Fujita likes to play. They found that Mahjong gives cells a competitive advantage, as—for example—in Drosophila mahj−/− cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. In mammals, Mahjong function is associated with cancer development and progression (Schabla et al. 2019). In the experiments that Prof. Fujita showed to us—using an MDCK cell line system similar to described above—the Mahjong expression was reduced by siRNA knockdown (Fig. 1B). He showed us a beautiful movie in which Mahjong knockdown cells (marked by GFP fluorescence) underwent apoptosis/blebbing in an MDCK cell line epithelial layer (Tamori et al. 2010 Plos Biology) of which a few screenshots are shown in Fig. 3. Importantly, if all the cells were Mahjong knockdown cells, apoptosis did not happen. Thus, Mahjong knockdown cells are “loser” cells if together with normal cells. Different from the Ras mutant cells, the Mahjong knockdown cells first died and only then were apically extruded (Tamori et al. 2010 Plos Biology).
Fig. 4 shows a summary by Prof. Fujita of the observed effects in cell competition induced by changes in various genes known to affect cancer development (cell transformation). In all these examples, the changed cells “lose,” either by apical extrusion as living cells or by apoptosis. The apical sides of epithelia are usually bordering a lumen that is—ultimately—associated with secretion from the body, so this direction of extrusion is thought to be a way to get rid of the cells.
Effect of RasV12 expression on cell competition within epithelia in vivo; a high fat diet promotes tumor formation
Prof. Fujita also investigated the effect on epithelial cell competition of RasV12 expression in vivo, by expressing it in only some of the cells in an epithelial monolayer of the small intestine in mice. To explain in detail: They used LSL-RasV12-IRES-EGFP mice (in which RasV12 expression can be induced in a Cre-dependent fashion) which was crossed with cytokeratin 19 (CK19; epithelial-specific marker)-Cre-ERT2 mice. In the hybrid mice, an appropriate low dose of tamoxifen (which activates the Cre recombinase complex and so removes the stop codon that prohibited the expression of RasV12) induced RasV12 expression in only some of the cells, leading to a mosaic of cancerous cells between normal cells.
Intriguingly, four days after the induction of RasV12 + GFP expression, in control mice in which GFP was expressed alone, the number of GFP-positive cells was much higher than in the mice that expressed GFP together with RasV12. This concluded that RasV12 expression induced the expressing cells to be actively eliminated. This elimination was shown to be predominantly through apical extrusion, as this was >25-fold higher than the basal extrusion (Kon et al. 2017).
The above system was also used to investigate the effect of RasV12 expression on cell competition in epithelia of other tissues in the mouse. Moreover, Prof. Fujita investigated several conditions for their influence on RasV12 cell elimination. For example, a mouse model for obesity (by high fat diet or “HFD”) showed reduced apical extrusion of the RasV12 cells in the epithelia of pancreatic ducts and intestine, which implies a greater chance to develop cancer (Sasaki et al. 2018 Cell Reports). In the pancreas, one month after induction of RasV12 expression, in the normally fed mice the cancerous cells were almost completely eliminated but in the HFD (obese) mice they formed a tumorous mass (Sasaki et al. 2018) (Figs. 5 and 6). Prof. Fujita assumes that these differences are caused by metabolic changes and chronic inflammation in the obese model. The finding agrees with obese people having a higher risk for developing cancer (CDC information).
What happens at the sites of competition during Epithelial Defense Against Cancer (EDAC)?
Prof. Fujita has named the processes by which normal epithelial cells eliminate cancerous cells in their midst Epithelial Defense Against Cancer (EDAC). So, what are the molecular mechanisms of EDAC? To find EDAC markers, Prof. Fujita and his group grew normal and transformed epithelial cells alone or as mixtures and compared the proteomes, phosphorylated proteins, and/or transcriptomes to look for clues (Kajita et al. 2014; Sato et al. 2020). Molecules that they found increased in the mixed culture are vimentin, plectin, and filamin. By immunocytochemistry they found that the vimentin was especially high in the normal cells at the contact sites with the cancerous (Src transfected) cells (Kajita et al. 2014) (schematically shown in Fig. 7).
He showed a beautiful movie revealing how the normal surrounding MDCK cells actively move and hereby “squeeze” the cancerous cell (MDCK-v-Src) out of their midst (screenshots in Fig. 8); even “poking” (protrusion) phenomena can be observed (screenshots in Fig. 9) (the movies are from Kajita et al. 2014). The normal cells that do not border the cancerous cell also move but this does not lead to any cell extrusion. In the movie, the non-transformed cells express GFP-vimentin, and the green signal (from GFP) is especially strong at the sites of contact with the transformed cell.
So, the extrusion seems to be a combination of normal epithelial processes, like the (slow) moving of the cells, and something “special” like the enhanced concentration of vimentin in the non-transformed cells at the contact site. It is unknown if the latter is a result of the reduction or absence of a normal epithelial intercellular signal (like would there also be an enhanced vimentin concentration if there would be a gap in the monolayer?) or the presence of a unique signal from the cancerous cell.
When vimentin was knocked down in these MDCK cells, the ratio of apical extrusion of the transformed cell was reduced more than two-fold. Vimentin is believed to participate in cytoskeleton formation in the normal cell at the border with the cancerous cell in a cascade of intracellular interactions (Kajita et al. 2014).
Effect of cancerous mutations in both Scribble and Ras
It seems to be an amazing system, the way that epithelia can get rid of cancerous cells. However, people do get tumors, so what happens? It is commonly agreed that mutations in multiple genes are usually required for a cancer to become “successful” (see also the summary of the presentation by Prof. Hans Clevers). Prof. Fujita applied this idea to cell competition in epithelial layers, asking the question of whether an additional mutation could change the cancer cells from “losers” into “winners.”
So, they checked what happened if the transcript for the tumor suppressor molecule Scribble was knocked down in all MDCK cells of a monolayer, and RasV12 was expressed in a few of them (Kohashi, Mori, Narumi et al. 2021). Essentially, they created “double mutant” Scribble-KD/RasV12 cells surrounded by “single mutant” Scribble-KD cells. Intriguingly, this led to a reversal of the direction of extrusion, with now the majority of Scribble-KD/RasV12 cells protruding from the basal side of the monolayer and only a minority extruding from the apical side (Fig. 10). In addition, they observed that surrounding single-mutant cells often were dying and gobbled up by the double-mutant cells (Fig. 10). Again, Prof. Fujita showed beautiful movies about this, which you can find easily accessible in Kohashi, Mori, Narumi et al. 2021. The authors often observed that a protrusion from the double-mutant cells made contact with a surrounding single-mutant cell and appeared to induce the death of the latter by apoptosis; they called this a “death-touch” (screenshots in Fig. 11; movie S3 in Kohashi, Mori, Narumi et al. 2021). In this system, the apoptosis in the cells surrounding the double mutants was >5-fold higher than in a control, and >80% of the double-mutant cells appeared to have engulfed surrounding single-mutant cells.
Then Prof. Fujita and his group checked, using the same system, the importance of apoptosis for the engulfment. Intriguingly, they found that blocking apoptosis by a caspase inhibitor did not lead to a reduction in the engulfment of the single mutant cells by the double mutant cells. In this case, however, the engulfment appeared to be of living instead of dead cells, which is a process named “entosis” (for entosis see wikipedia and hms.harvard.edu) (Kohashi, Mori, Narumi et al. 2021).
Interestingly, only if surrounded by single mutant (RasV12) cells, the double mutant (Scribble-KD/RasV12) cells detectably express the phagocytic marker CD68 and not if surrounded by similar double mutant cells (Kohashi, Mori, Narumi et al. 2021).
Future
Prof. Fujita stressed that we still do not know well about the intercellular recognition pathways involved in cell competition. It is possible that various types of differences are recognized. Currently, Prof. Fujita is very interested in the physical properties of the cells, the importance of which is already indicated by the above-described squeezing mechanism. He also mentioned that it would be good to have markers for cell competition so the process can be more easily followed and investigated. Most importantly, it would be nice if we could find out methods that stimulate our bodies to fight developing cancers by EDAC and learn to avoid conditions (such as obesity) that inhibit EDAC.
