皮膚癌におけるKM48モノクローナル抗体反応性CD44変異体の役割の研究

2014 Fiscal Year Research-status Report

• Project/Area Number: 26860904
• Research Institution: Kurume University
• Principal Investigator: TEYE KWESI 久留米大学, 皮膚細胞生物学研究所, 研究員 (30599303)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: KM48 / CD44 / squamous cell carcinoma / epidermis / skin

Outline of Annual Research Achievements

The purpose of this project is to identify the variant of CD44 recognized by KM48 antibody and to assess its functional role in normal and human skin cancer by various methods. Expression of more than 800 transcripts of CD44 is possible but not all are expressed and the expression is tissue and context dependent. To date, however, the nature of these isoforms in human epidermis has not been adequately determined. For the first time, all CD44 transcripts in human epidermis were isolated and analyzed in details. It was found that human epidermis express a total of 18 transcripts, and 3 new ones were found. To provide a clue as to which of these transcripts are associated with development of epithelial cancer such as squamous cell carcinoma (SCC) and because KM48 expression is lost in SCC, regulation of expression of CD44 transcripts was analyzed and compared between normal and malignant keratinocytes. It was found that expression of CD44v8-10 was high in malignant cells as compared to normal keratinocytes. It was also found that expression of CD44v2-10 was high in normal epidermal keratinocytes but was low in cultured malignant keratinocytes. Furthermore, normal and malignant keratinocytes were found to produce different CD44 transcripts upon serum stimulation and subsequent serum starvation, suggesting that specific CD44 isoforms are involved in tumorigenesis via different CD44-mediated biological pathways.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

Analysis of CD44 is complicated by its complex alternative splicing. It takes time and much effort to isolate all CD44 transcripts in a given tissue. Isolation of all CD44 transcripts is also hampered by differential expression of transcripts with different sizes and different level of expression. The breakthrough happened when a novel cloning strategy was developed to successfully isolate, clone, analyze and designate all 18 CD44 transcripts in human epidermis. It was worthwhile to study the regulation of CD44 transcripts by various agents to identify CD44 transcripts and proteins that may segregate wit oncogenic and mitogenic activities in cultured normal and malignant keratinocytes. Overall, the project running smoothly but the plan had to be adjusted or modified to accommodate unforeseen variations in experimental design and results.

Strategy for Future Research Activity

Now that all CD44 transcripts in human epidermis and skin were isolated and designated, it is now possible to design specific assays to examine the presence and fates of these transcripts in normal and disease states. Using these cDNA clones, the variant of CD44 recognized by KM48 antibody will be identified under specialized cell culture conditions including 3-D cell culture. Because the KM48 antibody is of IgM class, its use for analysis is difficult. It became necessary to convert the IgM to IgG from by cloning the variable region from the hybridoma cells and joining it to the constant region of mouse IgG. This will be used as tool to analyze the KM48 protein in various experimental settings including Western blotting, immunofluorescence and most importantly immunoprecipitation of the KM48 protein from epidermal extract as well as cultured cell extracts. The IgG version may also be better suited for peptide mapping to identify the CD44 variant recognized by the KM48 antibody. The rest of the projects concerns with performing functional studies using KM48 antibody on cells. Both the IgM and IgG versions of KM48 antibody will be added to cells and cell behavior such as migration, adhesion, differentiation, proliferation and desmosome assembly will be investigated.

Causes of Carryover

Real time PCR master mix and primers for Real time PCR to compare the transcription level of CD44 between normal and malignant skin tissues has not yet been purchased due to a slight delay in obtaining these tissues from patients.

Expenditure Plan for Carryover Budget

The project is still on going and the remaining amount of money will be used to purchase the following items: Real time PCR master mix and primers.

Research Products

• [Presentation] Isolation of all CD44 transcripts in human epidermis and regulation of their expression by various agents (2014)
  • Author(s): Kwesi Teye, Sanae Numata, Rafal P. Krol, Atsunari Tsuchisaka, Takahiro Hamada, Tadashi Karashima, Chika Ohata, Minao Furumura, Marek Haftek, Norito Ishii, Takashi Hashimoto
  • Organizer: 39th Annual Meeting of the Japanese Society for Investigative Dermatology
  • Place of Presentation: Hotel Hankyu Expopark, Suita-City, Osaka
  • Year and Date: 2014-12-12 – 2014-12-14