CD133 in Disease

Photoreceptor Degeneration

Despite the expression of CD133 in a wide range of tissues ranging from stem cells to differentiated cells of all germ layers, the most striking phenotype of loss- of-function mutation is disk dysmorphogenesis and retinal dystrophy in the eye197,236_{. Photoreceptor cells are part of the retina, a light-sensitive layer of} tissue able to transform light into nerve signals, which can be interpreted by the brain.

CD133 is expressed in both photoreceptor cells, namely cones and rods. The latter is concerned with vision in dim light whereas cones are involved in vision in bright light as well as colour perception. In rods, CD133 is specifically expressed at the base where newly synthesised membrane evaginations or disks develop from the connecting cilium and move up the tract197,237_{. In cones, CD133 is}

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observed in the outer segment along the whole tract236_{. Interestingly,} immunoreactivity of CD133 is different in rods versus cones. The epitope AC133 which is often used as a human stem cell marker is mainly detected in the outer segment of cones whereas CD133 in rods was detected using αhE2, an antiserum216_{. This might indicate that different forms of CD133 are present in} photoreceptors.

To date, five mutations in the human PROM1 gene responsible for retinal dystrophy affecting both cones and rods have been discovered. Most mutations lead to the introduction of a premature stop codon, and all of them share many symptoms including but not limited to night blindness, gradual loss of peripheral vision and photoreceptor degeneration197,238–242_.

Studies in mice replicating loss-of-function of CD133 gave the first clues as to what the function of CD133 is. CD133 KOmice displayed an age-dependent loss of photoreceptors. Indeed, by the age of one year, the entire photoreceptor layer of mice lacking CD133 was missing resulting in full blindness236_{. Mice carrying} the human R373C PROM1 gene have abnormally shaped disk membranes and excessively large outer segments by P16. At four months, photoreceptor disks were either misaligned and were of excessive size compared to wild-type mice or defects in outer segment membrane organisation241_{. Remarkably, eyecup} cultures treated with cytochalasin-D, an inhibitor of actin polymerisation, display a similar phenotype. Other studies showed that non-functional CD133 leads to defects in new disk formation, i.e. disks protruding from the ciliary plasma membrane. The interaction between actin, CD133 and Protocadherin 21 (PCDH21) as shown by co-immunoprecipitation might be crucial for the proper formation of new disks. Indeed, PCDH21 and CD133 are both present in newly-

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made disks and in R373C mice non-functional CD133 leads to mislocalisation of PCDH21 and its impaired proteolytic cleavage197,241,243_{. The role of actin with the} CD133-PCDH21 complex is also not clear but might be important in the proper distribution of the complex in a temporospatial manner241_{. The localisation of} CD133 in the outer segment of disk evaginations might prevent fusion of disks as is evident from the separation of disks in cones.

Tumour-initiating cells

The traditional model of carcinogenesis states that any dividing cell of the body can give rise to a tumour if enough mutations that permit unlimited proliferation potential occur in its genome. This dogma was overturned in the late 90’s by the discovery of cells within myeloid lymphoma244 _{that possess stem cell features} and were capable of generating the typical heterogenic population seen in many tumours245_{. Later, these stem cell-like tumourigenic cells were also discovered in} solid tumours. Expansion of mammary stem cells in mouse breast cancer models prior to cancer development represent a probable link between normal stem cells and tumour-initiating cells also called cancer stem cell246_{. Mainly these cells have} been identified through the expression of cell-specific surface markers. Among the most frequently shared markers by tumour-initiating cells of different germlines are CD20, CD24. CD34, CD44, CD90, CD117, CD133, Aldehyde dehydrogenase (ALDH), nestin, β1-integrin and Epithelial cell adhesion molecule (EpCAM)247_.

One of the most commonly used tumour-initiating cell markers across different tumour types is CD133. Singh et al. took CD133+ cells from human brain tumours and was able to show that they exhibit stem cell-like features, by proving they were capable of self-renewal and replication of an original tumour when grafted

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into an immunodeficient brain of NOD/SCID mice with as little as 100 cells. Injection of 100 CD133- cells showed no tumorigenic response248_{. Confirming this} result, Chiou et al., revealed that in atypical teratoid rhabdoid tumours, a malignant tumour of the central nervous system, the metastasis, malignancy and radioresistant capabilities of CD133+ were substantially increased when compared to CD133- cells249_.

Interestingly, Rappa et al. investigated CD133 as a possible therapeutic target for metastatic melanoma. Retroviral-mediated knockdown of CD133 in FEMX-1 melanoma cells showed slower growth and reduced capacity to form spheres compared to control cells. This suggests that the cells have lost some of their oncogenic properties. Knockdown of CD133 severely reduced the capacity of the cells to metastasise indicating that CD133 may have a role in the capacity of tumour cells to migrate to other tissues250_{. Indeed, immunohistochemical analysis} of human hepatocellular carcinoma samples (HCC) showed that the number of CD133+ CD44+ HCC cells is increased and linked with portal vein invasion251_. This was partially confirmed by Horst et al. showing that high levels of CD133 correlate strongly with synchronous liver metastasis, but knockdown of the receptor indicated no change in colony formation or proliferation252_{. Contrary to} these findings, different groups have expressed their concerns regarding CD133 as a tumour-initiating cell marker. Using IL10-/-_CD133LacZ/+ _{mice, Shmelkov} showed that both CD133+ and CD133- metastatic colon cancer cells were capable of long-term tumorigenesis in immunodeficient mice and that the latter formed larger tumours94_{. Interestingly, the same study showed that CD133} expression in adult CD133LacZ/+ _{mice is not restricted to stem cells but is also} present in luminal terminally differentiated cells such as but not restricted to the pancreas and kidney. Several studies have confirmed this work claiming that

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expression of CD133 in colon cancer is not restricted to tumour-initiating cells 253_. Likewise, in healthy cells of the gastrointestinal tract CD133 is uniformly expressed throughout the tissue94,254_{. Ogden et al. and Yoshikawa et al. revealed} that CD133 could be a biliary and progenitor cell marker in vivo, concluding that CD133 alone is not sufficient to detect tumour-initiating cells in human hepatocellular carcinoma cell lines255,256_{. Therefore, the validity of CD133 being} an oncogenic marker is still controversial.

Another major therapeutic problem tumour-initiating cells present is their resistance to treatment such as radiation and various chemotherapy, which generally target homogeneous populations of rapidly proliferating, differentiating tumour cells. Alternative targets are being looked at. These include the drug Salinomycin, a potassium ionophore that can reduce the proportion of CD133+ subpopulations in human colorectal cancer cell lines HT29 and SW480. Furthermore, Salinomycin treatment decreases colony-forming ability and cell motility in HT29 cells and decreases vimentin expression and induces E-cadherin expression in HT29 cells257_.

It is, therefore, imperative to try to understand the mechanisms contributing to this resistance which can only be achieved by identifying the cancer stem cells, the signalling pathways and their regulatory mechanisms involved in tumour- initiating cell maintenance. As discussed earlier, radiation and chemotherapy resistance, as well as limited cell numbers, has been a major therapeutic problem for combating CD133+ tumour-initiating cells.