HDGF was shown to be overexpressed in several types of tumors, like e.g. hepatocellular carcinoma [
17,
18] or melanoma [
23]. The correlation between the degree of HDGF overexpression, disease prognosis [
16,
18,
20‐
22,
24] and the role for HDGF in tumor angiogenesis [
25,
26], metastasis [
19,
26], and apoptosis [
27,
28] suggested that HDGF may function as an oncogene/protooncogene. In this study, a mouse model was generated to address the question whether HDGF-overexpression mediates oncogenic/transforming capacity to non-transformed cells
in vivo. We chose to target the HDGF expression to melanocytes with the help of a Tyrosinase promoter/enhancer element (Figure
1A) since results presented by Bernard and co-workers revealed HDGF-expression in melanomas whereas HDGF was absent or weakly present in nontumorigenetic melanocytes [
23]. In addition, HDGF-expression was graded with progression, suggesting that the frequency of HDGF-expression increases from benign nevi gradually to late melanoma stages [
23]. Therefore, if HDGF exhibits oncogenic/transforming capacity on melanocytes, loss or overexpression of HDGF are expected to have an influence on tumor development and/or progression.
Transgene induced HDGF overexpression in melanocytes could be confirmed by comparing melanocytes and tissues of wildtype and HDGF
Tyr/HDGF
-/- mice (Figure
2, Additional file
1). No alterations regarding melanocyte number and localization could be observed
in vivo in HDGF
Tyr, wildtype and HDGF
-/- mice, which were additionally analyzed to investigate whether the HDGF-deficiency has an impact on melanocytes in the further conducted experiments. To favor the development of tumors we introduced a second genetic alteration and applied an additional mutagenic noxa. This was done by deleting one copy of the Ink4a tumor suppressor gene and exposure to mutagenic doses of UV-light. Neonatal sunburn of HDGF
Tyr/Ink4a
+/-, Ink4a
+/-, and HDGF
-/-/Ink4a
+/- mice led to the development of pigmented skin abnormalities (Figure
3). These abnormalities, however, did not resemble melanocytic lesions, but strongly resembled human epidermoid cysts. These cysts are benign, round or oval in shape, are in contact with the skin surface via a pore, and are confined by a squamous epithelium which, through cornification, produces a perl of keratinized material [
41]. Epidermoid cysts arise most frequently from the infundibulum of hair follicles and represent a keratinocyte differentiation defect. Pigmentation of epidermaoid cysts follows a definite anatomic pattern and is dependent in humans on the natural skin color [
42,
43]. No differences regarding pigmentation of murine epidermoid cysts of HDGF
Tyr/Ink4a
+/-, Ink4a
+/-, or HDGF
-/-/Ink4a
+/- mice could be observed. Therefore, melanocytes located at the squamous epithelium of epidermoid cysts seem to derive from follicular melanocytes and the observed increased number of melanocytes may be due to growth factors released from cells of the benign cysts. Detailed analysis of size and number of epidermoid cyst per animal revealed a difference between the genotypes. Thus, HDGF
-/-/Ink4a
+/- mice developed significantly more and larger epidermoid cysts compared to both HDGF
Tyr/Ink4a
+/- and Ink4a
+/- mice (Figure
3A-C). Morphologically keratinocytes of the outer root sheat (ORS) resemble keratinocytes of the basal cell layer with which they form a continuous cell layer. The morphology of epidermoid cysts indicate that ORS keratinocytes run through the differentiation program of epidermal keratinocytes [
44] resulting in the formation of a perl of keratinized material. It is not clear why Ink4a
+/- mice develop epidermoid cysts after a single neonatal UVB-treatment, but our results show that additional HDGF-deficiency leads to more and larger epidermoid cysts. The presented results therefore indicate that HDGF in wildtype mice is involved in the control of keratinocyte differentiation. So far, HDGF was reported to promote differentiation of e.g. vascular smooth muscle cells [
5,
45,
46]. In addition, Enomoto and coworkers recently reported on the investigation of an albumin-HDGF transgenic mouse model targeting HDGF-overexpression to hepatocytes [
47]. In this case the overexpression of HDGF resulted in delayed hepatocyte maturation, suggesting that HDGF-overexpression in hepatocytes partially suppresses hepatocyte differentiation. In contrast, delayed melanocyte maturation in HDGF
Tyr mice was not obvious since skin and hair pigmentation of neonatal HDGF
Tyr mice was indistinguishable from wildtype littermates. The only hint on an impact of elevated HDGF-levels on melanocyte differentiation came from
in vitro experiments. When we tried to culture primary melanocytes from HDGF
Tyr, wildtype and HDGF
-/- newborn mice, HDGF
Tyr melanocytes were not able to differentiate and grow in a confluent pigmented cell layer (Figure
2B).
In conclusion, the results obtained here implicate that HDGF does not convey an oncogenic/transforming capacity to melanocytes. In fact, our results and those from other groups point to an involvement of HDGF in cell differentiation. Nevertheless, the overexpression of HDGF in various tumors and its validity as a prognostic marker in some tumors points to a significant role of this growth factor. Over the last years, the concept of non-oncogene addiction (NOA) was established. This concept describes the fact that tumors depend on the expression or overexpression of several proteins, which are not oncogenes, but which are secondarily regulated and thus promote tumor progression [
48]. These proteins, like e.g. Stat5 or HSF1, help tumor cells to overcome cellular stress which in normal cells would lead to apoptosis [
49,
50]. In case of HSF1-deprivation tumor cells die and tumors regress whereas normal cells do not depend on this protein [
51]. The latter certainly applies for HDGF as shown by the normal development and health of HDGF
-/- mice [
15]. On the other hand,
in vitro and
in vivo experiments showed that a reduction of HDGF in transformed cells or the injection of HDGF specific antibodies resulted in slowed tumor growth, reduced number of blood vessels, and increased rate of apoptosis [
25,
52‐
54]. These results and the results of this study strongly suggest that HDGF does not possess oncogenic/transforming capacities. Instead our results and the results from other groups made us realize that HDGF exhibits many criteria to fit into the concept of non-oncogene addiction. This recommends HDGF as a potential drug target with relevance for a variety of tumors.