A majority of human cancers originate from epithelial tissue. A common cancer of epithelial origin is nonmelanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), with more than 700,000 new cases diagnosed each year in the United States. Similar cancers are also seen in non-human animals such as domesticated animals and pets, including cats and dogs. BCC is rarely life-threatening because it is slow growing and is mostly localized. Unlike BCC, SCC metastasizes at a rate of 2% to 6% over several years after initial diagnosis. A highly malignant form invades and destroys tissue, and then metastasizes, initially to a regional lymph node before more distant organs such as the lung or brain are affected. SCC is commonly encountered in a number of epithelial tissues, including the oral cavity, esophagus, larynx, bronchi, intestines, colon, genital tract, and skin. Early detection using reliable biomarkers is desired, as are rationally designed drugs for effectively preventing and treating aggressive, metastatic SCC.
As such, there is a need for a good animal model system for studying how metastatic squamous cell carcinoma develops, progresses and can be treated. To date, no such model exists. Classically, tumor cells are injected into the tail vein of either immunocompromised or syngeneic mice. While this assay can suitably model the later metastatic stages, it does not model the early genesis, invasion and angiogenic stages of malignant progression, especially as it relates to complex interactions between tumor and host, especially at the tissue site where the carcinoma originated. Moreover, the role of the immune system in metastatic progression cannot be analyzed when immunocompromised mice are used.
Murine skin model systems are still essential contributors to our understanding of the multi-step nature of chemically-induced carcinogenesis. In the multistage mouse skin carcinogenesis model, biochemical events unique to initiation, promotion, or progression can be studied and related to cancer formation. In that model, the NMSC that is most often induced is squamous cell carcinoma. Although squamous cell carcinoma of mouse skin invades the dermal region, the incidence of malignant metastatic conversion is rare and requires a long latency period of approximately a year.
Several protocols are used to develop mouse skin tumors in laboratory animals. In a common initiation-promotion protocol, mouse skin is treated with an initiating agent (7,12-dimethylbenz[a]anthracene; DMBA) and then with a potent tumor promoter (12-O-tetradecanoylphorbol-13-acetate; TPA). In this protocol, mice develop mostly benign papillomas, more than 90% of which regress after TPA treatment is stopped. Only a small percentage of papillomas progress to invasive, but non-metastatic, SCC. The initiation-promotion protocol has been further modified to enhance the conversion of skin papillomas to carcinomas, yet metastatic potential is not increased.
A major intracellular receptor for TPA is the ubiquitous enzyme protein kinase C (PKC), an important signal transduction pathway component for controlling cell proliferation and tumorigenesis. It has been suggested that PKC activation may play a role in promoting mouse skin tumor formation. However, several groups have demonstrated that repeated applications of TPA depress PKC activity and protein levels. These results indicate that both loss of PKC activity and degradation of PKC could be important for mouse skin tumor promotion by TPA.
On the basis of the structural similarities and cofactor requirements, the eleven known PKC isoforms are grouped into three subfamilies: (1) the conventional PKCs (xcex1, xcex2I, xcex2II, and xcex3), which depend upon Ca2+, phosphatidylserine (PS), and diacylglycerol (DAG) or TPA; (2) the nPKCs (xcex5, xcex4, xcex7, and xcex8), which require only PS and DAG/TPA; and (3) the atypical PKCs ("igr"/xcex and "xgr"), which retain PS dependence but have no requirement for Ca2+ or DAG/TPA for activation. PKCxcexc, which is usually classified as a nPKC, is not easily grouped with any of the other isoforms.
The roles of PKCxcex1 and PKCxcex4 isoforms in the mouse skin tumor initiation/promotion protocol were assessed in FVB/N transgenic mice expressing an T7-epitope-tagged PKCxcex1 (T7-PKCxcex1) or PKCxcex4 (T7-PKCxcex4) under the control of the human keratinocyte-specific K14 promoter/enhancer. Transgenic expression of T7-PKCxcex1 did not affect tumor promotion susceptibility. Transgenic expression of T7-PKCxcex4 in the epidermis (xcx9c8-fold increase) suppressed the formation of both skin papillomas and carcinomas by 70%.
PKCxcex5 may play an important role in cellular growth regulation. TPA binds to and activates PKCxcex5. Activated PKCxcex5 may be important for the survival of small cell lung carcinoma cell lines in which the catalytic fragment of PKCxcex5 is constitutively expressed. Overexpression of PKCxcex5 in Rat-6 or NIH-3T3 fibroblasts increases growth rate, anchorage independence, and tumor formation in nude mice. PKCxcex5 overexpression also transforms non-tumorigenic rat colonic epithelial cells and suppresses apoptosis of interleukin-3 dependent human myeloid cells induced by removal of interleukin-3.
The role of PKCxcex5 in mouse skin tumor promotion and epidermal cell growth and differentiation remains unclear. Treatment of the mouse skin with TPA leads to a general reduction in PKC activity that persists for at least 4 days. Acute TPA treatment decreases PKCxcex2 and xcex7 protein levels, but has little or no effect on the levels of PKCxcex1, xcex4, or xcex5. PKCxcex1, xcex2, and xcex4 activity levels were reduced after acute or repeated TPA treatments, but PKCxcex5 activity was not examined. DMBA/TPA-induced papillomas exhibit decreased cytosolic levels of PKCxcex1 and xcex2II protein, but insignificant alterations in the levels of PKCxcex4, xcex5, or "xgr" protein. When cultured mouse skin keratinocytes are induced to differentiate by increasing Ca2+, PKCxcex5, xcex4, and a translocate to the membrane fraction, suggesting a role for activation of these isoforms in keratinocyte differentiation.
The present invention is summarized in that an FVB/N mouse that expresses more PKCxcex5 in its epidermis than in the epidermis of a wild-type FVB/N mouse is a useful model for development and treatment of skin cancer, particularly squamous cell carcinoma, in human and non-human mammalian animals. In a preferred embodiment, the mouse expresses at least about 5-fold more epidermal PKCxcex5 than wild-type FVB/N mice, with more preferred embodiments having still higher levels of epidermal PKCxcex5.
The present invention is also summarized in that an FVB/N mouse that expresses PKCxcex5 in its epidermis at a level higher than a wild-type FVB/N mouse, where the level is sufficiently high to induce metastatic growth, is a useful model for development and treatment of metastatic skin cancer, particularly for metastatic squamous cell carcinoma, in humans. Notably the mice constitute a model system for developing and treating highly malignant metastatic squamous cell carcinoma. A level sufficiently high is more than 5-fold higher than in wild-type FVB/N mice, and is preferably at least about 12-fold higher, and still more preferably at least about 15-fold higher, and most preferably at least about 18-fold higher.
The present invention is also summarized in that a method for inducing squamous cell carcinomas in the aforementioned mice includes the steps of treating the mouse with a skin tumor initiating chemical agent, then treating the mouse repeatedly with an skin tumor promotion chemical agent for a time sufficient to induce squamous cell carcinomas and then screening the treated mice to identify those mice in which squamous cell carcinoma is induced. In a related embodiment, the skin tumor initiating agent can be DMBA and the skin tumor promotion agent can be TPA. After treatment according to the method, the mice of the invention are characteristically suppressed for papilloma formation, even though squamous cell carcinoma is observed at an enhanced rate. The mice having squamous cell carcinoma disease produced in the inducing method are also an useful animal model for development and treatment of squamous cell carcinoma induced by non-chemical agents such as ultraviolet radiation.
In a related aspect, the present invention is further summarized in that a method for inducing squamous cell carcinomas in FVB/N mice that express PKCxcex5 in its epidermis at the aforementioned level sufficiently high to induce metastatic growth consists essentially of the step of treating the mice with an initiating agent without further treatment with a promoting agent.
In a related embodiment, the present invention is summarized in that a method for inducing metastatic moderately differentiated squamous cell (MDSC) carcinomas in an FVB/N mouse that expresses at least about 12-fold more PKCxcex5 in its epidermis than a wild-type FVB/N mouse includes the steps of treating the mouse with a skin tumor initiating agent and then treating the mouse repeatedly with an skin tumor promotion agent for a time sufficient to allow metastatic involvement, typically, but not exclusively, of the lymph nodes. The metastatic MDSC thus induced appear to originate from the hair follicle within squamous cells located near the sebaceous gland (xe2x80x9cbulge regionxe2x80x9d) that are postulated to be progenitor or stem cells for the hair follicle and epidermis. The metastatic MDSC are pathologically distinguishable by histogenesis from the well differentiated squamous cell carcinomas (WDSC) observed in FVB/N mice having a wild-type PKCxcex5 level that appear to originate instead from the interfollicular epidermis and which invade the dermis and subcutaneous tissues, but remain localized.
In a related aspect, the invention is summarized in that a method for evaluating the effectiveness of putative agents against squamous cell carcinoma in a mammal includes the steps of administering various doses of at least one putative agent over an appropriate range to mice of the invention that have squamous cell carcinoma, evaluating the effect of the agent on development or progression of the squamous cell carcinoma, and selecting at least one agent having anti-squamous cell carcinoma activity.
The invention is still further summarized in that a method for evaluating the effectiveness of putative agents against metastatic squamous cell carcinoma in a mammal includes the steps of administering at least one putative agent in varying amounts to mice of the invention having metastatic squamous cell carcinoma, evaluating the effect of the agent on development or progression of the metastatic squamous cell carcinoma, and selecting at least one agent having anti-metastatic squamous cell carcinoma activity.
The present invention is also summarized in that a mouse that expresses at leas about 5-fold more PKCxcex5 in its epidermis than a wild-type FVB/N mouse can be made by increasing the rate at which a PKCxcex5-encoding polynucleotide is transcribed, by decreasing the rate at which a transcript of the PKCxcex5-encoding polynucleotide is degraded, or by increasing the stability of the transcript or of a resulting PKCxcex5 protein in epidermal cells.
In a related aspect, the present invention is summarized in that a method of making a mouse that expresses at least about 5-fold more PKCxcex5 in its epidermis than a wild-type FVB/N mouse includes the steps of introducing into a one cell fertilized FVB/N embryo a chimeric transgene that comprises a polynucleotide that encodes PKCxcex5 under the transcriptional control of an upstream promoter active in keratinocytes and a downstream polyA addition sequence, implanting the embryo in a carrying animal, screening progeny of a cross between offspring of the carrier animal and FVB/N mice for expression of the transgene for PKCxcex5 expression level, and selecting transgenic offspring having increased PKCxcex5 expression.
The present invention is yet further summarized in that a transgene for use in the method of making a mouse that expresses at least about 5-fold more PKCxcex5 in its epidermis than a wild-type FVB/N mouse comprises a polynucleotide that encodes PKCxcex5 under the transcriptional control of an upstream promoter active in keratinocytes and a downstream polyA addition sequence, and optionally includes a polynucleotide that encodes a peptide tag adjacent to the polynucleotide that encodes PKCxcex5 and further optionally includes one or more other transcription enhancing elements.
It is an object of the invention to provide a non-human animal model system for cutaneous squamous cell carcinoma and metastatic squamous cell carcinoma in other human and non-human mammalian animals.
It is another object of the invention to provide an animal model system where the non-human animal develops an aggressively malignant and metastatic disease.
It is a feature of the present invention that carcinomas induced in the mice of the invention in a two-stage tumor initiation/promotion method suppress the formation of skin papillomas and enhance the formation of moderately differentiated squamous cell carcinomas.
It is another feature of the present invention that when the level of PKCxcex5 is sufficiently high in mice of the invention, moderately differentiated squamous cell carcinomas of the animals rapidly metastasize to regional lymph nodes, there by mirroring cutaneous metastatic squamous cell carcinoma disease in humans and other non-human mammalian animals.
It is an advantage of the present invention over existing murine model systems in that the invention permits study of metastatic development in a timely manner.
It is yet another advantage of the present invention that the carcinogen is administered topically.
It is yet another advantage of the present invention that the carcinomas appear rapidly, within 15 to 25 weeks, thus facilitating its use in screening for agents that can prevent induction of metastatic SCC and as a model for investigating the genesis and progression of SCC, and the molecular events associated with progression and metastasis.
Other objects, advantages and features of the invention will become apparent upon consideration of the following detailed description.
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