E2F modulates keratinocyte squamous differentiation: implications for E2F inhibition in squamous cell carcinoma

E2F regulation is essential for normal cell cycle progression. Therefore, it is not surprising that squamous cell carcinoma cell lines (SCC) overexpress E2F1 and exhibit deregulated E2F activity when compared with normal keratinocytes. Indeed, deliberate E2F1 deregulation has been shown to induce hyperplasia and skin tumor formation. In this study, we report on a dual role for E2F as a mediator of keratinocyte proliferation and modulator of squamous differentiation. Overexpression of E2F isoforms in confluent primary keratinocyte cultures resulted in suppression of differentiation-associated markers. Moreover, we found that the DNA binding domain and the trans-activation domain of E2F1 are important in mediating suppression of differentiation. Use of a dominant/negative form of E2F1 (E2F d/n) found that E2F inhibition alone is sufficient to suppress the activity of proliferation-associated markers but is not capable of inducing differentiation markers. However, if the E2F d/n is expressed in differentiated keratinocytes, differentiation marker activity is further induced, suggesting that E2F may act as a modulator of squamous differentiation. We therefore examined the effects of E2F d/n in a differentiation-insensitive SCC cell line. We found that treatment with the differentiating agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or expression of E2F d/n alone had no effect on differentiation markers. However, a combination of E2F d/n + TPA induced the expression of differentiation markers. Combined, these data indicate that E2F may play a key role in keratinocyte differentiation. These data also illustrate the unique potential of anti-E2F therapies in arresting proliferation and inducing differentiation of SCCs.     

Modulation of proliferation-specific and differentiation-specific markers in human keratinocytes by SMAD7

We examined the potential role of SMAD7 in human epidermal keratinocyte differentiation. Overexpression of SMAD7 inhibited the activity of the proliferation-specific promoters for the keratin 14 and cdc2 genes and reduced the expression of the mRNA for the proliferation-specific genes cdc2 and E2F1. The ability of SMAD7 to suppress cdc2 promoter activity was lost in transformed keratinocyte cell lines and was mediated by a domain(s) located between aa 195-395 of SMAD7. This domain lies outside the domain required to inhibit TGFbeta1 signaling, suggesting that this activity is mediated by a novel functional domain(s). Examination of AP1, NFkappaB, serum response element, Gli, wnt, and E2F responsive reporters indicated that SMAD7 significantly suppressed the E2F responsive reporter and modestly increased AP1 activity in proliferating keratinocytes. These data suggest that SMAD7 may have a role in TGFbeta-independent signaling events in proliferating/undifferentiated keratinocytes. The effects of SMAD7 in differentiated keratinocytes indicated a more traditional role for SMAD7 as an inhibitor of TGFbeta action. SMAD7 was unable to initiate the expression of differentiation markers but was able to superinduce/derepress differentiation-specific markers and genes in differentiated keratinocytes. This latter role is consistent with the ability of SMAD7 to inhibit TGFbeta-mediated suppression of keratinocyte differentiation and suggest that the opposing actions of SMAD7 and TGFbeta may serve to modulate squamous differentiation.     

Ca2+ and BMP-6 signaling regulate E2F during epidermal keratinocyte differentiation

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E2F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell Growth Differ. 8, 553-563). Thus, we hypothesized that various E2F proteins likely play distinct growth regulatory roles in the undifferentiated stem cells and in terminally differentiated keratinocytes. To further understand the function of E2F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E2F factors in undifferentiated cultured murine primary keratinocytes or in cells induced to differentiate with Ca(2+) or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E2F regulation with both differentiating agents and demonstrate a switch in expression from E2F-1, -2, and -3 in undifferentiated, proliferating cells to E2F-5 in terminally differentiated keratinocytes. Inhibition of keratinocyte proliferation by transforming growth factor-beta1 did not enhance E2F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E2F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E2F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E2F5 specifically inhibited DNA synthesis in undifferentiated keratinocytes in an HDAC-dependent manner, suggesting that E2F-5.p130.HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of keratinocytes responding to differentiation stimuli.     

The enzymatic activity of lysyl oxidas-like-2 (LOXL2) is not required for LOXL2-induced inhibition of keratinocyte differentiation

Lysyl oxidase-like-2 (LOXL2) induces tumor progression and fibrosis. It also inhibits the differentiation of keratinocytes promoting development of squamous cell carcinomas. Stimulation of HaCaT skin keratinocytes with exogenous LOXL2 or overexpression of LOXL2 in these cells inhibits their differentiation as manifested by inhibition of calcium or vitamin D-induced involucrin expression. The inhibition was abrogated by the LOXL2 function-blocking monoclonal antibody AB0023 as well as by an anti-LOXL2 polyclonal antibody. Surprisingly, a point-mutated form of LOXL2 (LOXL2(Y689F)) lacking enzymatic activity, as well as a LOXL2 deletion mutant lacking the entire catalytic domain, also inhibited calcium or vitamin D-induced up-regulation of involucrin expression, suggesting that the enzymatic activity of LOXL2 is not required for this activity. This conclusion was supported by experiments that showed that β-aminoproprionitrile, an irreversible competitive inhibitor of the enzymatic activity of all lysyl oxidases, is unable to abolish the LOXL2-induced inhibition of HaCaT cell differentiation. The activity of LOXL2(Y689F) required the presence of the fourth scavenger receptor-cysteine-rich (SRCR) domain of LOXL2, which is also the binding target of AB0023. Epitope-tagged LOXL2(Y689F) was internalized at 37 °C by HaCaT cells. The internalization was inhibited by AB0023 and by competition with unlabeled LOXL2, suggesting that these cells may express a LOXL2 receptor. Our results suggest that agents that inhibit the enzymatic activity of LOXL2 may not suffice to inhibit completely the effects of LOXL2 on complex processes that involve altered states of cellular differentiation.     

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1.