Induction of KLF4 in mouse skin
As transgenic animals carrying constitutive keratin promoters exhibited lethality, we used alternate inducible and constitutive strategies to direct KLF4 to basal cells. KLF4 was linked to a tetracycline response element (TRE) (), and seven founder lines were crossed to mice carrying a keratin 14 (K14)-reverse tetracycline-responsive transcriptional activator (rtTA) transgene (). This X chromosome-linked K14-rtTA
was previously shown to direct expression of TRE-linked transgenes to K14-positive cell types (Xie et al., 1999
). Induction with doxycycline (dox) yielded a skin phenotype in male animals of two lines, 32831 and 32812. No phenotype was observed in K14-rtTA
control animals treated with dox. The results shown below were obtained using 32831 mice, carrying an autosomal TRE-KLF4
, although similar results were obtained using 32812 (not shown). A phenotype in TRE-KLF4
females required homozygosity of the K14-rtTA
Figure 1 Doxycycline (dox) inducible KLF4 transgenic mouse lines. (a) A transgene composed of the keratin 14 (K14) promoter and the reverse tetracycline-responsive transcriptional activator (rtTA) directs expression of tetracycline response element (TRE)-linked (more ...)
To detect human KLF4 transcripts, we utilized the size difference of mouse and human products of reverse transcriptase (RT)-PCR obtained using a pair of conserved primers (, upper panel). Expression of transgene-derived transcripts was comparable to that of endogenous KLF4 at two days (lane 3). By 21 days, a majority of the transcripts were derived from the transgene (lane 5). No product was obtained without addition of RT (lanes 2, 4, and 6), indicating successful removal of genomic DNA from the samples. Detectable transgene expression required both the rtTA (middle panel, lanes 3, 5) and dox (upper panel, lane 1). As for whole skin, Northern analysis of RNA from primary keratinocyte cultures revealed undetectable levels of transgene expression without dox (). Expression was readily detected after 3 hours of induction, and gradually increased until 24 hours.
KLF4 rapidly induces dysplasia and SCC-like skin lesions
Following 2 days of dox treatment, skin morphology was indistinguishable from uninduced animals or control mice (). In contrast, at 9 days the skin showed hyperkeratosis, atrophy of the sebaceous glands, cystic degeneration of hair follicles, and hyperplasia (). After 3–4 weeks, we observed crusted skin lesions and moistness and thinning of the pelage of the ventral skin, with hyperplastic, keratinizing epithelium extending into the dermis (). Dysplastic changes included loss of basal cell polarity and delayed maturation of squamous epithelium. By 42 days lesions resembled severe dysplasia or SCC in situ, with hyperchromatic, pleomorphic nuclei and mitotic figures (). Extension of cells into the dermis were similar to superficially invasive SCC, but lacked the invasive property of fully malignant cells and did not efface the adjacent skeletal muscle layer.
Figure 2 Histology of the skin following induction of KLF4. (a–f) Dox was administered for the indicated interval and ventral skin of male animals was analyzed. The dermo-epidermal junction (DEJ) is marked with a dashed line (c, e). In parallel with these (more ...)
Progression of dysplastic lesions observed at 21 days to SCC-like lesions required continued induction. Withdrawal of dox on days 21–42 lead to resolution of the phenotype (). RT-PCR revealed no expression of the transgene following withdrawal (not shown).
KLF4 is predominantly nuclear in mouse skin lesions and human cutaneous SCC
mRNA in situ
hybridization analysis detected KLF4 in dysplastic mouse epithelium, but not in adjacent mesenchymal cells, consistent with restriction of transgene expression to K14-positive cell types, as previously reported for this K14-rtTA
line (Xie et al., 1999
)(). KLF4 antibody stained a subset of nuclei in this epithelium, and stained more diffusely within the cytoplasm (). Little or no staining was observed in normal mouse skin (), perhaps because only part of the immunizing peptide is conserved.
We previously showed that KLF4 is upregulated in virtually all cases of HNSCC (Foster et al., 1999
; Foster et al., 2000
). To determine if KLF4 is expressed in cutaneous cancers, we stained 5 cases of SCC and one case of basal cell carcinoma (BCC). Two of the SCCs exhibited prominent nuclear staining, in contrast to weak staining of adjacent epithelium (). Three other SCCs and the BCC showed little or no staining, indicating that KLF4 is differentially regulated in skin tumors.
Dysplastic lesions exhibit similarities with SCC
To molecularly characterize the lesions, we analyzed the cytokeratins K14, K1, K16, and K17, and the proliferation marker PCNA in ventral skin (). K14, normally confined to the basal cell layer (, No dox), stained basal and many parabasal cells after 2 days of induction. By 9–21 days nearly all the epithelial cells were K14 positive.
Figure 3 Immunostaining of KLF4-induced lesions. The indicated antibodies were applied to sections of ventral skin of males. Arrowheads indicate the DEJ. Asterisks indicate lesions deeper within the dermis that are PCNA-positive and K1-low, similar to human SCC. (more ...)
Prior to induction, PCNA was prominent in basal cells of the interfollicular skin, but low in parabasal cells (, No dox). As for K14, PCNA was rapidly induced in parabasal cells of interfollicular skin (, days), and largely mirrored K14 at later timepoints. PCNA persisted in basal and parabasal cells within spheres of epithelial cells deep within the dermis (, PCNA, 21 days, asterisks).
K1, a marker of early differentiation in interfollicular cells (, No dox), was rapidly induced by KLF4 in outer root sheath keratinocytes of the hair follicle, indicating an alteration of cell fate (, days). At day 9, K1 was expressed in the majority of epithelial cells, but was later restricted to more differentiated, suprabasal cells, and was largely negative in cells deeper in the dermis (, 21 days, asterisks). Analysis of skin at 21 days using the follicle marker K17 revealed uniform staining of cystic follicles, but not of the dysplastic surface epithelium, consistent with derivation of dysplastic epithelium from both cell types (not shown).
K16 was low prior to induction as expected, but focally positive in basal keratinocytes of interfollicular epithelium by 2 days (). At 9 days, expression was uniform in interfollicular cells, and focally positive in follicular cysts. K16 was prominent in suprabasal cells at day 21.
In summary, KLF4 induced outgrowth of dysplastic, squamous epithelial lesions composed of K14-, PCNA-, and K16-positive cells that gradually lost K1, similar to human cutaneous SCC (van der Velden et al., 1997
; Horn and Bravo, 1998
). Rapid induction of K14 and PCNA in parabasal cells at 2 days is consistent with inhibition of the proliferation-differentiation switch that normally occurs in developing epithelium.
KLF4 induces an apoptotic response
Induction of KLF4 in cultures of bitransgenic, primary keratinoctyes resulted in death of the vast majority of cells by 48 hrs (not shown). To determine whether KLF4 can induce apoptosis in vivo, we analyzed frozen sections by TUNEL. Prior to induction, TUNEL-positive squamous cells were rare (). By day 2 parabasal cells exhibited staining. Apoptosis peaked at day 9, when both interfollicular cells and follicular cysts were strongly positive. In contrast, staining of severely dysplastic skin was less frequent (, day 38). Thus, KLF4 induces a transitory apoptotic responses that diminishes in association with progression to dysplasia.
Figure 4 TUNEL analysis of apoptosis following induction of KLF4. Frozen sections were labeled with digoxigenin (dig)–dUTP and terminal transferase. Dig was detected using an alkaline phosphatase conjugated antibody and the substrate Fast Red (arrowheads). (more ...)
p53 deficiency promotes KLF4-induced dysplasia
The established role of p53 in the genesis of SCC suggested that deficiency of this tumor suppressor could promote the KLF4-induced phenotype (Boyle et al., 1993
; Kemp et al., 1993
; Ziegler et al., 1994
; Jonason et al., 1996
; Brash and Ponten, 1998
; Jonkers et al., 2001
). We identified settings in which p53 deficiency was important or essential for KLF4-induced dysplasia ( and ). Unlike p53 wild-type males (p53+/+
), in which the phenotype was largely confined to the ventral skin (see above), induction of KLF4 in p53 hemizygous knockout animals (p53+/−
) induced pronounced gross and microscopic changes of both the dorsal and ventral skin (not shown). Whereas the dorsal skin of p53+/+
males exhibited only focal, mild dysplasia (, left panel), p53+/−
animals similarly exhibited severe dysplasia in association with infiltration of the dermis by a well-vascularized, cellular and fibrotic stroma, or fibrovascular response (FVR) (, middle and right panels). No dysplasia was observed in p53-deficient mice in the absence of KLF4 induction.
Figure 5 p53 gene dosage alters the skin phenotype of TRE-KLF4 transgenic mice. (a) Analysis of dorsal skin in p53 wild-type (p53+/+) or deficient (p53+/−, p53−/−) males. p53+/+ animals exhibited only focal, minor involvement (left panel, (more ...)
Figure 6 Analysis of MMTV-KLF4 transgenic mice. (a) p53+/− animals developed dorsal hair loss, dysplasia, and fibrotic skin between 6 and 8 mo. of age in association with subcutaneous sarcoma. Dysplasia appeared similar to the inducible model, with a prominent (more ...)
The above studies utilized males, in which our expression strategy is expected to yield relatively uniform induction. However, human SCCs are clonally derived from small clusters of cells (Brash and Ponten, 1998
; Mao et al., 2004
). To examine whether induction of KLF4 in a mosaic pattern could lead to dysplasia, we examined females, in which random inactivation of the X chromosome leads to expression of X-linked, hemizygous alleles in patches measuring less than 1.0 mm (Deamant and Iannaccone, 1987
; Ng et al., 1990
; Gao et al., 2002
). In K14-rtTAX+/−;TRE-KLF4+/−;p53+/+
females, mosaic KLF4 was insufficient to induce any gross phenotype. By microscopy there were only focal changes including cystic follicles and mild changes in the adjacent interfollicular epithelium (e.g., , left panel). However, the dysplastic phenotype was consistently observed in females that were either p53+/−
(, middle panel) or K14-rtTAX+/+
(, right panel). These results suggest that KLF4 must be expressed in a sufficient patch size to induce dysplasia, and that a smaller patch size is sufficient in p53-deficient skin.
Constitutive KLF4 induces dysplasia in a p53-dependent fashion
To determine the consequence of longer term expression of KLF4, we utilized the mouse mammary tumor virus promoter (MMTV) promoter, which is active in skin (Hennighausen et al., 1995
; Jonkers et al., 2001
). As no phenotype was observed by 18 months of age, we crossed the p53
knockout allele into each of four lines. By 8 months of age, MMTV-KLF4;p53+/−
animals of two lines developed dorsal skin lesions similar to those observed in the inducible model (above), with dysplastic skin overlying a vascular, fibrotic dermis (100% of 15 animals; ). Ten p53+/−
littermate controls showed no abnormality by 8 months of age (Fisher’s exact test, P<0.001). Likewise, MMTV-KLF4;p53+/−
animals at 3–6 months of age exhibited no phenotype (). Thus, progression from grossly normal skin to dysplasia occurred between 6 and 8 months of age in MMTV-KLF4;p53+/−
The life-span of these animals was limited to 8 mo. due to the outgrowth of sarcoma within the dysplasia-associated FVR (100% of 15 animals) (not shown). These 1.0–1.5 cm diameter tumors were composed of spindled, anaplastic cells and scattered giant cells. KLF4, p63, or cytokeratin antibodies did not stain sarcoma cells, but did stain the overlying dyplastic epithelium (). The FVR may lead to sarcoma when additional defects are acquired in the responding p53+/− mesenchymal cells, consistent with the known predisposition of p53−/− animals to this neoplasm.
Three MMTV-KLF4;p53+/+ animals that were >18 months of age developed severe dysplasia of the dorsal skin or ears, with delayed maturation, nuclear pleomorphism and hyperchromicity (). Although sarcoma was not observed, a FVR included blood vessels intercalated between basal and parabasal epithelial cells (). The skin phenotypes and the associated mesenchymal changes observed in KLF4 transgenic mice are summarized in .
Role of expression pattern and p53 status in KLF4-induced dysplasia.
Localization to the nucleus correlates with transforming activity
Although KLF4 can function in the nucleus to regulate the activity of several cellular or viral promoters (Jenkins et al., 1998
; Zhang et al., 1998
; Mahatan et al., 1999
; Shie et al., 2000
; Zhang et al., 2000
; Hinnebusch et al., 2004
), mechanistic insight into its role as an oncogene is lacking. To identify molecular changes following acute activation of KLF4, we generated a 4-OHT conditional allele by fusion of KLF4 to a portion of the estrogen receptor (KLF4-ER) (Littlewood et al., 1995
). Upon addition of 4-OHT to culture media, the fusion protein underwent rapid translocation to the nucleus (). To test for transforming activity, we used a volume of KLF4-ER retroviral supernatant sufficient to transduce only a small fraction of cells (i.e., <1.0%), as indicated by resistance to puromycin. When 4-OHT was included in the growth media, KLF4-ER induced transformed foci on a background monolayer of wild-type RK3E cells (). The spindled, refractile appearance of cells composing these foci were typical of KLF4-transformed cells (Foster et al., 1999
; Pandya et al., 2004
). Few or no foci were observed in the absence of 4-OHT, indicating the strictly conditional nature of the allele.
Figure 7 Characterization of KLF4-ER. (a) KLF4 was fused to a 4-hydroxytamoxifen (4-OHT)-responsive fragment of the mouse estrogen receptor (ER). Stably-transduced cells were stained to enable localization of KLF4-ER. Nuclear localization results in a pink color (more ...)