Patent 6,897,352

Non-human mammalian animals having a higher epidermal expression level of protein kinase Cε than their wild-type counterparts are phenotypically distinguished from wild-type animals in that the animals induced to develop tumors in a chemical initiation/promotion protocol are suppressed for subsequent papilloma development but are susceptible to developing squamous cell carcinoma and metastatic squamous cell carcinoma. The animals are advantageously used in methods for screening putative agents for altering the susceptibility, development and progression of squamous cell carcinoma and metastatic squamous cell carcinoma and have further commercial value as tools for investigating the development of metastatic disease.

Description of the Invention

BACKGROUND OF THE INVENTION

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 (α, βI, βII, and γ), which depend upon Ca2+, phosphatidylserine (PS), and diacylglycerol (DAG) or TPA; (2) the nPKCs (ε, δ, η, and θ), which require only PS and DAG/TPA; and (3) the a typical PKCs ι/λ and ζ), which retain PS dependence but have no requirement for Ca2+ or DAG/TPA for activation. PKCμ, which is usually classified as a nPKC, is not easily grouped with any of the other isoforms.

The roles of PKCα and PKCδ isoforms in the mouse skin tumor initiation/promotion protocol were assessed in FVB/N transgenic mice expressing an T7-epitope-tagged PKCα (T7-PKCα) or PKCδ (T7-PKCδ) under the control of the human keratinocyte-specific K14 promoter/enhancer. Transgenic expression of T7-PKCα did not affect tumor promotion susceptibility. Transgenic expression of T7-PKCδ in the epidermis (˜8-fold increase) suppressed the formation of both skin papillomas and carcinomas by 70%.

PKCε may play an important role in cellular growth regulation. TPA binds to and activates PKCε. Activated PKCε may be important for the survival of small cell lung carcinoma cell lines in which the catalytic fragment of PKCε is constitutively expressed. Overexpression of PKCε in Rat-6 or NIH-3T3 fibroblasts increases growth rate, anchorage independence, and tumor formation in nude mice. PKCε 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 PKCε 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 PKCβ and η protein levels, but has little or no effect on the levels of PKCα, δ, or ε. PKCα, β, and δ activity levels were reduced after acute or repeated TPA treatments, but PKCε activity was not examined. DMBA/TPA-induced papillomas exhibit decreased cytosolic levels of PKCα and βII protein, but insignificant alterations in the levels of PKCδ, ε, or ζ protein. When cultured mouse skin keratinocytes are induced to differentiate by increasing Ca2+, PKCε, δ, and α translocate to the membrane fraction, suggesting a role for activation of these isoforms in keratinocyte differentiation.

Chronic exposure to UV radiation in sunlight is an important risk factor for non-melanoma epidermal carcinogenesis and for other common recurrent skin injuries such as sunburn and premature cutaneous photoaging. The UV spectrum, having wavelengths between those of visible light and x-rays, is conventionally divided into three major wavelength groups: UVA (315-400 nm), UVB (280-315 nm) and UVC (190-280 nm). UVA and UVB are the most prominent and ubiquitous carcinogenic wavelengths, as ozone in the stratosphere absorbs most of the radiation below 310 nm. The UVA and UVB components are highly genotoxic, but do not penetrate deeper than the skin. The biological effects mediated by UVA and UVB are subtly different.

UV radiation is considered to be a complete carcinogen because it both initiates and promotes carcinogenesis. UVB induces oxidants that directly damage DNA and induce gene mutations, the key initiating genetic events in carcinogenesis. Gene mutations associated with UV-induced skin cancer include TP53, PITCH and ras oncogenes. UV-induced oxidants may also regulate a protein kinase network that controls gene expression associated with skin tumor promotion. UV radiation also potently suppresses the immune system.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized in that an FVB/N mouse that expresses more PKCε 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 PKCε than wild-type FVB/N mice, with more preferred embodiments having still higher levels of epidermal PKCε.

The present invention is also summarized in that an FVB/N mouse that expresses PKCε 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 PKCε 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 PKCε 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 ("bulge region") 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 PKCε 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 least about 5-fold more PKCε in its epidermis than a wild-type FVB/N mouse can be made by increasing the rate at which a PKCε-encoding polynucleotide is transcribed, by decreasing the rate at which a transcript of the PKCε-encoding polynucleotide is degraded, or by increasing the stability of the transcript or of a resulting PKCε 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 PKCε 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 PKCε 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 PKCε expression level, and selecting transgenic offspring having increased PKCε 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 PKCε in its epidermis than a wild-type FVB/N mouse comprises a polynucleotide that encodes PKCε 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 PKCε and further optionally includes one or more other transcription enhancing elements.

The invention is also summarized in that an FVB/N mouse that expresses PKC_ε in its epidermis at a level higher than a wild-type FVB/N mouse is more sensitive than wild-type FVB/N mice to skin damage induced by UV irradiation. Even after a single exposure of UVB light (6 kj/m²), the mice suffered skin damage that did not heal normally. Moreover, such irradiated mice exhibited substantial epidermal hyperplasia and mutation in p53. Both hyperplasia and mutation in p53 characteristically precede photoaging and skin cancer.

It is an object of the invention to provide an 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 an object of the present invention to provide a model system for investigating mechanisms of UV-induced skin damage.

It is another object of the invention to provide hypersensitive animals useful in a method for screening compounds for preventing or treating photodamage and photoaging of skin.

It is yet another object of the invention to provide an animal resistant to wound healing for use in a method for screening possible wound healing agents.

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 PKCε 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.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a murine model system for development and progression of squamous cell carcinomas, and more particularly a model system for development and progression of metastatic carcinomas and methods for making and using mice for evaluating agents for modulating development and progression of metastatic carcinomas, as well as the mice per se. Certain aspects of this invention are described in Reddig, P. J. et al., "Transgenic Mice Overexpressing Protein Kinase Cε in Their Epidermis Exhibit Reduced Papilloma Burden but Enhanced Carcinoma Formation after Tumor Promotion," Cancer Research 60:595 (2000), incorporated herein by reference as if set forth in its entirety.

Mice within the scope of the invention are characterized as FVB/N mice that express PKCε at or above about five times the level seen in wild-type FVB/N mice. The effects that result from elevated epidermal PKCε levels vary as the epidermal level increases. Overexpression of PKCε in the untreated mouse epidermis leads to phenotypic abnormalities (such as inflamation, hyperkeratosis, hyperplasia, cellular hypertrophy, and ulceration), especially of the skin surrounding the tail base of older mice (approximately 7-8 months of age).

Of particular note in the disclosed system is the different character of the tumors induced in mice of the invention after a tumor initiation/promotion regimen. The mice of the invention are suppressed for papilloma formation and instead develop moderately differentiated squamous cell carcinomas (MDSC) without requiring papillomas as a precursor. The MSDC appear to originate from the hair follicle within squamous cells located near the sebaceous gland ("bulge region"). The bulge cells are thought to be progenitor or stem cells for the hair follicle and epidermis. Interestingly, after treatment certain mice of the invention also develop papilloma-independent squamous cell carcinoma that metastasizes rapidly to the lymph nodes when the activity level of PKCε in the epidermis is particularly high, say, at least about 12-fold higher than the wild-type level, or more preferably greater than 15 fold higher, most preferably about 18 fold higher.

In contrast, wild-type FVB/N mice treated in the same regimen typically first develop papillomas and then well differentiated squamous cell carcinomas (WDSC) that invade the dermis and subcutaneous tissues but remain localized. The WDSC largely appear to originate from the interfollicular epidermis.

The mice and methods of the invention provide a unique opportunity to study the origin and events associated with malignant progression (and interruption thereof) and have direct commercial value to the biotechnology community as a result. Separately, the mice and methods provide a resource with which to evaluate the role of PKCε in tumorigenesis. The difference in metastatic potential and the different origin of malignancy support the conclusion that T7-PKCε papilloma-independent carcinomas are pathologically distinct from the mouse carcinomas seen in treated wild-type FVB/N mice.

Mice having elevated epidermal PKCε activity levels in accordance with the invention can be produced by increasing the rate at which a PKCε-encoding polynucleotide sequence is transcribed, by decreasing the rate at which the transcript is degraded, or by increasing the stability of the transcript or of the resulting PKCε protein in epidermal cells. One suitable and convenient method for increasing the PKCε level in epidermal cells is to provide in the cells a transgene that comprises a known PKCε coding sequence flanked upstream by a promoter effective in the target skin cells and downstream by a suitable polyA sequence, preferably from the same source as the promoter sequence. Other elements that can be provided on the transgene include transcription enhancing sequences, such as a β-globin intron, or sequences that can aid in detecting the encoded PKCε protein, such as a T7 tag that can be readily detected using a commercially available horseradish peroxidase-conjugated anti-T7-tag antibody (Novagen, Inc., Madison, Wis.). Preferably, the additional sequences should not negatively affect the expression or activity of the PKCε protein.

Nucleotide sequences for the components of a suitable transgene are attached in the Sequence Listing. A K14 promoter/enhancer sequence from humans is attached as SEQ ID NO:1. A suitable P-globin intron is attached as SEQ ID NO:2. A PKCε encoding polynucleotide sequence from mice is attached as SEQ ID NO:3. At the 5′ end of SEQ ID NO:3 is a T7 tag sequence. SEQ ID NO:4 depicts the encoded PKCε polypeptide sequence. SEQ ID NO:5 is a K14 poly A addition sequence from humans. The skilled artisan will appreciate that the functions of the disclosed sequences are not adversely affected by certain mutations in the aforementioned polynucleotide and amino acid sequences, including insertions, deletions or substitutions, including but not limited to changes that result in conservative changes to any resulting amino acid sequence. Moreover, some changes in either the PKCε coding sequence or a regulatory sequence can increase the PKCε activity in the epidermis and such changes are specifically contemplated to be within the scope of the invention.

The transgene can be provided to epidermal cells or to cells that mature into epidermal cells, such as keratinocytes or keratinocyte precursor cells. Most preferably, the transgene is inserted into an early stage fertilized embryo in a method for making transgenic mice. The embryo is allowed to mature in a manner known to the art. Introduction of the chimeric gene into the fertilized egg of the mammal is accomplished by any number of standard techniques in transgenic technology (Hogan et al., 1986, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor, N.Y.). Most commonly, the chimeric gene is introduced into the embryo by way of microinjection. The PKCε-encoding polynucleotide sequence under the control of the strong heterologous promoter can conveniently be introduced into the recipient mouse strain by microinjection of a chimeric expression cassette (or "transgene") into pronuclei, preferably male pronuclei, of 1-cell fertilized mouse embryos using known transgenic methods, as described below in connection with the working embodiment of the invention. Other methods, including classical breeding methods, can also be used to produce animals expressing elevated levels of PKCε in the skin of the animal.

It is important to perform the transgenic technique with a recipient mouse strain that is both susceptible to tumor formation and to transgenic uptake of exogenous DNA. Moreover, the mouse strain should be inbred to eliminate undesired and uncontrollable effects that can be introduced by heterozygosity. Mice suitable for use in the methods of the invention, including the method for producing transgenic mice, are FVB/N inbred mice (commercially available from Taconic Farms, Inc.) and closely related strains and derivatives thereof, although other mouse strains may also be suitable. The FVB/N inbred mouse strain produces eggs that have easily discernible pronuclei and produce large litters for an inbred strain. FVB/N mice are also sensitive to multistage carcinogenesis. See Taketo, M. et al., P.N.A.S. U.S.A. 88:2065-2069 (1991), incorporated by reference herein as if set forth in its entirety.

Animals obtained in the method can be screened for integration of the PKCε-encoding polynucleotide and for production of PKCε in the epidermis. A skilled artisan can use known methods to determine whether a transgene has been incorporated into the genome of offspring mice and can readily determine the level of expression of the transgene, for example either by probing for antibodies directed to a tag on the transgene or antibodies directed to the PKCε protein sequence itself. Preferred methods are described in the Example.

Skin tumors can be induced in the mice using a standard initiation-promotion regimen wherein a single dose of a tumor initiator (e.g., DMBA) is applied to the backs of the animals. Several weeks after initiation, a regimen of applying a tumor promoting agent (e.g., TPA) is undertaken. The time required to observe phenotypic changes in treated mice will vary somewhat with the mouse strain and with the level of PKCε expression in the mouse.

The mice of the invention having either squamous cell carcinoma disease or metastatic squamous cell carcinoma disease as described as useful for evaluating the effectiveness of one or more putative agents for treating the disease. For purposes of this invention, an agent is effective if it reduces the incidence or number of carcinomas on an animal or if reduces the severity of the carcinomas or if it reduces or eliminates metastatic disease in those animals in which metastatic disease is observed. In a suitable method for evaluating the effectiveness of a putative agent, the agent, either alone or with a plurality of other putative agents, is administered topically, orally, intraperitoneally, intramuscularly, or in any other suitable manner to an animal or animals having the disease to be treated in an amount to be determined on a case by case basis, but typically on the order of about 1 mg to about 1 gram (preferably in the range of between about 10 mg and about 100 mg) per mouse having a typical weight of about 25 grams. The effect on the animal of the treatment is then evaluated and those agents having an effect on the disease are selected. The agent(s) can have advantageous chemopreventative effect upon the development of metastatic disease in animals of the invention when administered in the same manner to the animal before or after development of squamous cell carcinoma in situ (pre-metastatic carcinoma). One can also screen for agents that reduce or reverse the progression of metastatic squamous cell carcinoma in human and non-human animals using the mice of the invention as a tester.

As a proof of concept, the inventors have demonstrated that when chemopreventative difluoromethylornithine (DFMO) was administered orally in drinking water to mice of the invention at between 50 and 100 mg per mouse, squamous cell carcinomas characteristic of the mice of the invention did not develop after initiation and promotion.

The FVB/N mice that produce a high level of PKC_ε in the epidermis are particularly useful in a method for screening batteries of compounds and selecting from the battery of compounds those compounds useful for preventing or reducing photodamage or photoaging, or for healing skin wounds. The mice are of particular value in that they are hypersensitized to the effect of radiation, thereby magnifying the effects of a compound that prevents or reduces photodamage or photohealing or heals skin wounds.

It will be understood that suitable compounds can be prophylactic or restorative. A prophylactic compound can be administered to a human or non-human animal before exposure to damaging UV radiation thereby preventing or reducing the stated effects of the radiation. A restorative compound can be administered after exposure to damaging UV radiation to undo the UV-induced photodamage or photoaging effects or to heal skin wounds in the animals.

In a method for screening compounds that prevent or reduce photodamage, the described FVB/N mice that exhibit the described sensitivity to UVB radiation are administered one or more compounds before or after receiving a skin-damaging dose of UVB irradiation (preferably in the range of 1 to 10 kj/m², typically about 2-8 kj/m², and preferably about 6-8 kj/m². Those compounds that reduce or prevent photodamage or photoaging effects or that heal skin wounds are identified.

Claim 1 of 7 Claims

1. A method for identifying an agent for reducing or preventing an effect of UV radiation on skin of a human or non-human animal, the method comprising the steps of:

exposing a genetically modified FVB/N mouse having epidermal cells that comprise a protein kinase Cε activity higher than that of wild-type FVB/N epidermal cells to an amount of UVB radiation sufficient to induce an effect selected from the group consisting of photodamage, photoaging, and skin wounding;

administering to the mouse at least one agent; and

determining whether the at least one agent can reduce or prevent photodamage, photoaging or skin wounding.

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