Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.
Summary Microphthalmia ‐ associated transcription factor (Mitf) is essential for melanocyte development and function and regulates anti‐apoptotic Bcl2 expression. We hypothesized that cellular deficiency of Mitf can influence melanocyte survival in response to ultraviolet (UV) radiation. Primary melanocyte cultures were prepared from neonatal wild‐type mice and congenic animals heterozygous for Mitf mutations Mitf mi‐vga9/+ and Mitf Mi‐wh/+ and exposed to UV irradiation. Wild‐type melanocytes were more resistant to UV‐induced apoptosis than melanocytes partially deficient in Mitf activity, as determined by relative levels of intracellular melanin and relative activation of Mitf target genes Tyr , Tyrp1, Dct, and Cdk2. Comparative experiments with wild‐type cells and congenic albino melanocytes demonstrated that these differences are not due to differences in melanin content, implicating Mitf as a primary determinant of UV‐dependent melanocyte survival. Mitf activity correlated directly with resistance to UV‐induced apoptosis in melanocytes. Mitf was important not only for regulating the expression of anti‐apoptotic Bcl‐2 following UV irradiation, but also the expression of the pro‐apoptotic BH3‐only Bad protein and activation of the extrinsic apoptotic pathway. Hence, Mitf is a multifaceted regulator of UV‐induced apoptosis in melanocytes.
Patients with the genetic disease type I neurofibromatosis (NF1) exhibit characteristic pigmentary lesions associated with loss of a single allele of NF1, encoding the 260 kDa protein neurofibromin. To understand the basis for these pigmentary problems, the properties of melanocytes haploinsufficient for the murine gene Nf1 were studied using Nf1+/– knockout mice. We demonstrate that neurofibromin regulates the Kit-Mitf signaling axis in vivo during melanocyte development. Primary Nf1+/– melanocytes were purified by FACS to measure melanogenic gene expression. We found that Nf1+/– melanocytes exhibit higher levels of melanogenic gene expression than their wild-type counterparts. Both prior to and following Kit stimulation, Nf1+/– melanocytes also exhibit increased activation of the MAP kinase pathway compared with primary cells. The melanogenic response of primary melanocytes to Mek inhibition is consistent with the changes observed with Nf1 haploinsufficiency; however, these changes differ from those observed with their immortalized counterparts. The observation that reduction of neurofibromin, either from haploinsufficiency in the case of primary melanocytes or from neurofibromin knockdown in the case of melan-a cells, enhances melanogenic gene expression suggests that neurofibromin plays a dominant role to MEK activity in controlling melanogenic gene expression in murine melanocytes.
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