This laboratory has shown previously that As+3
exposure can cause the direct malignant transformation of the UROtsa human urothelial cell line. This single isolate was shown to form subcutaneous tumor heterotransplants consistent with those of a urothelial carcinoma of the bladder [Sens et al. 2004
]. An additional interesting feature of these tumors was the presence of prominent areas of urothelial cells that had undergone squamous differentiation. As detailed in the introduction, a poor outcome has been associated with patients having urothelial carcinoma cells with squamous differentiation. Since the initial study was based on the generation of a single As+3
-transformed urothelial cell culture, the first goal of the present study was to determine the repeatability of the transformation event. Eight independent cultures of UROtsa cells were exposed to 1.0 μM As+3
following the identical protocol to that described previously by the laboratory [Sens et al. 2004
]. The successful isolation of a new As+3
-transformed transitional isolate was defined by its ability to form colonies in soft agar and subcutaneous tumor heterotransplants in immunocompromised mice. Employing this criteria, five new isolates were isolated that were shown to have the ability to form colonies in soft agar and form subcutaneous tumors in nude mice. The initial characterization of the 5 isolates showed very similar phenotypic properties to the initial isolate reported using the above protocol. The morphology of the cells at the light microscopic level of examination were very similar and all showed an epithelial morphology. An important feature was that each isolate produced a subcutaneous tumor that displayed a similar tumor histology and that this histology was similar to that expected for an in situ
human urothelial cell cancer. Furthermore, while the extent of the areas of urothelial cells showing squamous differentiation varied among each independent isolate, all displayed prominent areas of urothelial cell squamous differentiation. Thus, the 5 additional As+3
-transformed urothelial isolates had very similar phenotypic properties to that reported previously for the initial As+3
An important phenotypic difference between the isolates was discovered when the 6 As+3 -transformed isolates were assessed for the ability to establish peritoneal tumors following intraperitoneal injection of the cells. Two of the As+3 -transformed isolates were able to form hundreds of tumors within the peritoneal cavity, one isolate a modest number of peritoneal tumors, and three isolates no or only one peritoneal tumor. This finding is potentially important since it is known from patients with bladder cancer that they tend to spread locally, requiring the ability of tumor cells to colonize the peritoneal organs following escape from the bladder. Approximately 80% of high grade transitional cell cancers are invasive. Aggressive tumors may extend only into the bladder wall; however, the more advanced stages invade the adjacent prostate and seminal vesicles in males, and the ureters and retroperitoneum in both males and females, and some produce fistulous communications to the vagina or rectum. Approximately 40% of these deeply invasive tumors metastasize to regional lymph nodes. Hematogenous dissemination, principally to the liver, lungs and bone marrow, generally occur late in bladder cancer and only with highly anaplastic tumors. As such, the current isolates may be particularly valuable in defining the genotypes underlying the differences in the ability of the individual isolates to colonize local sites outside of the bladder. It should be noted that the ability of the cells to colonize the peritoneal cavity gives no information on the initial stages involved in metastasis, which includes invasion and escape from the bladder proper. However, the isolates could provide valuable information on the ability of cells that have escaped the local tumor environment and need to re-seed and grow at distant organ sites to complete the cycle of metastasis. Thus, the phenotypic differences among the As+3 -transformed isolates and their tumor heterotransplants should provide an excellent platform to define underlying genomic and proteomic differences that correlate with these individual phenotypes.
The ability of several of the As+3
-transformed isolates to form peritoneal tumors also allowed a comparison of tumor histology between the peritoneal and subcutaneous transplant sites. It was one of the goals of the present study to determine if the subcutaneous environment was one that favored prominent squamous differentiation of the urothelial cells. This was especially important in the present study since human exposure to arsenic is known to cause hyperkeratosis of the skin [Steinmaus et al. 2000
]. The results of this analysis clearly showed that peritoneal tumors displayed vastly reduced squamous differentiation compared to the subcutaneous transplant site. While areas of squamous differentiation could be found for each peritoneal tumor examined, the occurrence and degree of such differentiation was greatly reduced for the peritoneal tumors. The majority of individual tissue sections showed urothelial cells with no evidence of squamous differentiation. A fortuitous aspect of the intraperitoneal injection also provided an internal control to compare squamous differentiation between the peritoneal and subcutaneous tumors in the same mouse. There is leakage of tumor cells into the subcutaneous space during the intraperitoneal injections which results in a small subcutaneous tumor. These subcutaneous tumors displayed prominent squamous differentiation and tumor histology identical to those described initially when each isolate was tested specifically for subcutaneous tumor growth. The results show that peritoneal tumors produced from the two As+3
-transformed isolates have reduced squamous differentiation, but that areas of squamous differentiation of the urothelial cells can still be visualized on H&E examination by diagnostic pathologists. As detailed in the introduction there is evidence that squamous differentiation is an indication of tumors having a poor prognosis for the patient. Despite this association of squamous differentiation with a poor prognosis, there is limited or no information in the literature on the mechanism that promotes the malignant urothelial cell to undergo squamous differentiation. This is likely due to a lack of a urothelial cell model system that undergoes such differentiation either in vitro
or in vivo
. Thus, the present cell culture and heterotransplant model should provide a unique system to probe the mechanism underlying the ability of a malignant urothelial cell to undergo squamous differentiation.
The laboratory has shown that keratin 6 was overexpressed in subcutaneous tumor heterotransplants generated from the original As+3
-transformed UROtsa isolate [Somji et al. 2008
]. It was also shown in this report using immunohistochemistry, that a subset of archival specimens of human bladder cancer also displayed focal expression of keratin 6 protein. These findings have allowed the laboratory to hypothesize that keratin 6 might be a general marker for squamous differentiation in bladder cancer and a biomarker for bladder cancers arising from heavy metal exposure. Further evidence in support of the first hypothesis was generated in the current study. An immunohistochemical analysis of keratin 6 staining in the subcutaneous heterotransplants generated from the 6 As+3
-transformed isolates showed a strong correlation of keratin 6 staining with areas of squamous differentiation. No areas of squamous differentiation were found on pathologic examination that were not immunoreactive for keratin 6. Similarly, there was a corresponding absence of keratin 6 immunoreactivity in areas of urothelial cells that displayed no squamous differentiation. The evidence for a direct relationship between keratin 6 immunoreactivity and squamous differentiation was reinforced further in an examination of the peritoneal tumors. In these tumors, squamous differentiation was reduced, and keratin 6 staining was identified only in regions having or suspected of having squamous differentiation on H&E examination. In fact, keratin 6 immunoreactivity allowed one to focus on, and more easily identified, areas in the peritoneal tumors that had areas of squamous differentiation. It was also noted, that keratin 16, the known type I keratin pair for keratin 6 [Moll et al. 2008
], followed an identical pattern of immunoreativity to keratin 6, but with less intense staining. Thus, there was a strong correlation of keratin 6 immunoreactivity with areas of squamous differentiation in tumors derived from As+3
-transformed urothelial cells.
There is a limited amount of information on the expression of K6/16 in normal and malignant cells and tissues [Moll et al. 2008
]. In normal human epithelial tissues, keratin 6/16 expression has been reported in basal cells of the respiratory epithelium and in the suprabasal compartment of non-keratinizing stratified squamous epithelia [Moll et al. 2008
]. In agreement, the previous study from this laboratory showed that the K6/16 pair was not expressed in normal urothelium [Somji et al. 2008
]. In normal tissues, the K6/16 pair have been associated with various hyperproliferative epidermal disorders, suggesting these keratins may be molecular markers for hyperproliferative keratinocytes [Weiss et al. 1984
; Moll et al. 2008
]. They are not expressed in non-proliferating skin keratinocytes, but during skin wounding the K6/16 pair are rapidly induced at the wound edge before migration and regeneration begins to occur [Paladini et al. 1996
]. Once healing is complete, the expression of K6/16 is down-regulated to undetectable levels. Keratin 6 has been detected in the luminal cells of the embryonic mammary gland but not the mature gland [Grimm et al. 2006
]. Keratin 6 has been detected in cells of the prostate gland with high potential for proliferation and differentiation [Schmelz et al. 2006
]. Keratin 6/16 expression has not been previously associated with squamous differentiation in human urothelial cell cancer or in As+3
derived experimental tumors. In contrast, K6/16 have been shown to be strongly expressed in squamous cell carcinomas of different sites, preferentially in inner, maturing layers of tumor cell nests [Moll et al. 1982
]. A low expression of keratin 6 has been found in adenocarcinomas of the uterine cervix, but it was not reported to be associated with areas of squamous differentiation but rather with metaplasia [Smedts et al. 1993
]. An antibody that recognizes both keratin 5 and keratin 6 has been advanced as a possible adjunct in the diagnosis of breast cancer of basal character, but these studies do not indicate which keratin/s (5, 6 or both) are being expressed in the tumors [Livasy et al. 2007
; Siziopikou and Cobleigh, 2007
; Tischkowitz et al. 2007
]. It had been suggested in early reports that keratin 5, and not keratin 6, is the keratin usually being observed in immunostaining protocols of breast tumors [Otterbach et al. 2000
]. A unique aspect of the present study is that keratin 6/16 expression identifies only the subpopulation of malignant urothelial cells showing squamous differentiation.
The expression of keratin 6, 16 and 17 mRNA and protein were examined in the As+3 -transformed isolates and tumor heterotransplants. The results posed more questions for future study than immediate answers. Keratin 6 and 16 were expressed in all the As+3 -transformed isolates and heterotransplants. However, there was only a weak, if any, correlation between the levels of mRNA and protein expression in the individual isolates. This was also true between the isolates and the corresponding tumor heterotransplants. That this variability was not simply experimental error was suggested by the findings with keratin 17, where expression of mRNA and protein was very consistent among and between the isolates and tumor heterotransplants. This suggests that there may be a component of post-transcriptional regulation in keratin 6 and 16 expression. A particularly interesting finding was that only the keratin 6a gene was expressed in the As+3 -transformed cell culture isolates; where as, both the keratin 6a and keratin 6b genes were expressed in all the tumor heterotransplants. Another potentially significant finding was found during the confocal analysis of the intracellular organization of keratin 6 expression in the As+3 -transformed isolates. In the As#1, As#3 and As#4 isolates, keratin 6 was organized into strongly stained intermediate filaments in a majority of the cells and it was these isolates that were able to form peritoneal tumor heterotransplants. In contrast, the other isolates and the parent UROtsa cells displayed no such organization of keratin 6 and no peritoneal tumors were formed by these isolates. Keratin 16 and 17 organization was similar among all the isolates.