WO2009072120A1 - Use of zinc in combination with chemotherapy for treating cancer - Google Patents

Abstract

The present invention relates to methods for treating cancer using compositions comprising zinc, particularly zinc chloride, in combination with other anticancer agents, comprising administering the compositions to a subject in need thereof by any suitable route of administration other than topical administration.

Description

USE OF ZINC IN COMBINATION WITH CHEMOTHERAPY FOR

TREATING CANCER

FIELD OF THE INVENTION

The present invention relates to methods for treating cancer using compositions comprising zinc, particularly zinc chloride, and at least one anticancer agent, wherein the compositions comprising zinc are administered to a subject in need thereof by any suitable route of administration other than topical administration for treatment of skin cancer.

BACKGROUND OF THE INVENTION

The p53 oncosuppressor has a well-established role in protecting against cancer development. The human p53 gene is mutated in about 50% of human cancers. In addition, p53 is intrinsically unstable and mutations usually reduce further its stability and inhibit the oncosuppressing function of p53. Various types of stress activate the tumor suppression activity of p53, mostly at post-translational level, by a series of modifications, including phosphorylation among others.

In this regard, p53 phosphorylation at serine 46 (Ser46), which is a late event after DNA damage, was shown to be a necessary step for inducing apoptosis in response to severe DNA damage (Oda et al., Cell, 102: 849-862, 2000). The important role for p53Ser46 in specific induction of apoptosis has been also confirmed by studies using MEFs cells derived from Ser46Ala knock-in mice. It has been further shown that defects in Ser46 phosphorylation contribute to the acquisition of the p53 resistance in an oral squamous carcinoma cell line.

Other proteins that phosphorylate p53 are disclosed for example in US Patent No. 7,138,236. Specifically, ATM - a protein derived from a gene mutated in patients suffering from the human autosomal recessive disorder Ataxia-telangiectasia and ATR (TM-Rad3 -related) phosphorylate p53 at a number of specific sites. US 7,138,236 further states that it would be of an enormous clinical benefit to identify a compound able to modulate the interaction between ATR (ATM-Rad3-related) and p53. Zinc is a very potent chemical that deeply penetrates and kills tissue. Zinc is commonly used as the active ingredients in topical medicaments, applied mainly to the skin, for treating external infections. For example, zinc is one of the active ingredients in the Bacitracin Zinc-Polymyxin B Sulfate ointment, indicated for topical treatment of a variety of localized skin and eye infections and for the prevention of wound infections. Use of ointments and pastes comprising zinc, particularly zinc chloride, is also known for the topical treatment of skin cancer and melanoma. US Patent No. 6,558,694 discloses a unit dose packaging for treating skin cancer, moles, warts, keratoses, skin tumors and melanoma, comprising a zinc chloride paste as the active ingredient. Further disclosed are means for effectively administering to the skin holding the zinc chloride paste.

It was recently published, by the inventors of the present invention, that zinc supplementation to MCF7 breast cancer cells restores p53 transcription activity and drug-induced apoptosis (Puca et al., Exp. Cell Res. ePub: Oct. 28, 2008, the content of which is incorporated by reference in its entirety herein.). There is an unmet need to identify compounds that can induce or reestablish the tumor suppression activity of dysfunctional wild type p53.

SUMMARY OF THE INVENTION

The present invention relates to methods for treating cancer comprising administering to a subject in need thereof compositions comprising zinc, particularly zinc chloride, by any suitable route of administration other than topical administration and further administering at least one anticancer agents.

The present invention is based in part on the unexpected discovery that loss of tumor suppressor activity in response to absence of HIPK2 that phosphorylates p53 at Ser46 could be reversed by zinc supplementation in vitro and in vivo. Moreover, in the presence of zinc the structure of p53 undergoes conformational changes thereby becoming accessible to other known Ser46 kinases, such as, p38, PKCδ, and DYRK. This surprising finding opens new possibilities for the treatment of many types of cancers that retained wtp53. According to one aspect, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a composition comprising zinc, with the proviso that the route of administration is other than a topical route of administration, specifically, topical administration for the treatment of skin cancer, further comprising administering to the subject at least one anticancer agent.

According to one embodiment, administering the composition comprising zinc and the at least one anticancer agent results in one or more therapeutic effects selected from the group consisting of: reduction in tumor size, induction of apoptosis in a tumor and inhibition of tumor cell proliferation.

According to another embodiment, the composition comprises a pharmaceutically acceptable salt of zinc. According to yet another embodiment, the composition comprises zinc chloride.

According to yet another embodiment, the composition is administered via a route selected from a group consisting of: oral, parenteral, rectal and by inhalation.

According to yet another embodiment, the composition is administered via a route selected from a group consisting of: intratumoral and intralesional, for other than skin cancers.

According to yet another embodiment, the subject in need thereof is human.

According to yet another embodiment, the cancer comprises cancer cells expressing wild type p53.

According to yet another embodiment, the cancer cells express low HIPK2. According to yet another embodiment, the cancer is selected from the group consisting of brain cancer, colon cancer, colorectal cancer, breast cancer, acute leukemia, lung cancer, kidney cancer, squamous cell cancer, testicular cancer, stomach cancer, melanoma, sarcomas, ovarian cancer, non-small cell lung cancer, esophageal cancer, pancreatic cancer, lymphoma, leukemia, neuroblastoma, mesothelioma, prostate cancer, bone cancer and heptocellular cancer.

According to yet another embodiment, each of the zinc and the at least one anticancer drug are administered via a different administration regime.

According to yet another embodiment, administering the at least one anticancer drug is selected from the group consisting of: administering the at least one anticancer drug prior to the administration of the composition comprising zinc, administering the at least one anticancer drug concurrent with the administration of the composition comprising zinc and administering the at least one anticancer drug following the administration of the composition comprising zinc.

According to an alternative embodiment, the composition comprises zinc further comprises the at least one anticancer drug.

According to some embodiments, the anticancer drug is selected from the group consisting of: alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors or antitumour agents

According to some embodiments, the anticancer drug is adriamycine. According to another aspect, the present invention provides use of zinc or a pharmaceutically acceptable salt thereof and at least one anticancer drug for the preparation of a medicament for treating cancer, with the proviso that the medicament is other than a topical medicament for the treatment of skin cancer.

According to yet another aspect, the present invention provides use of zinc or a pharmaceutically acceptable salt thereof for the preparation of a medicament, with the proviso that the medicament is other than a topical medicament for the treatment of skin cancer, for treating cancer in combination with at least one anticancer drug.

According to yet another aspect, the present invention provides a composition comprising zinc and at least one anticancer drug for treating cancer, with the proviso that the composition is other than a topical composition for the treatment of skin cancer.

These and further objects, features and advantages of the present invention will become apparent from the following detailed description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates the resistance of HIPK2 and p53 depleted cell lines to ADR- induced apoptosis.

Figure 2 is a presentation of DNA microarray analyses showing that p53 target gene transcription in response to ADR is compromised by knock-down of HIPK2 to a larger extent than p53 knock-down. Figure 3 ChIP analyses performed with p53 antibody on C-RKO (A) and HIPK2i (B) cells treated with ADR and reporter luciferase assay (C).

Figure 4 exhibits analysis of p53 subcellular distribution and phosphorylation in C- RKO and HIPK2i cells

Figure 5 exhibits tumor growth in HIPK2i-derived tumors compared to p53i-derived tumors.

Figure 6 demonstrates the switch of p53 folded/unfolded states and restoration of p53 binding to target gene promoters in HIPK2i cells following zinc supplementation. Figure 7 presents growth of HIPK2i-derived tumor, in vivo, under treatment with ADR and ZnCl₂.

Figure 8 is a Kaplan-Meier plot showing survival curves of colon cancer patients with respect to HIPK2 expression and p53 status.

Figure 9 presents selected genes upregulated in C-RKO cells after ADR treatment, compared to HIPK2i and p53i cells (see Figure 2A, B). Figure 10 shows selected genes out of the 85 upregulated in HIPK2i cells (see Figure 2A, C).

Figure 11 presents oligomers for semi quantitative RT-PCR.

Figure 12 shows oligomers for promoters amplification in ChIP analysis.

Figure 13 shows the effect of lentiviral LV-THsi/HIPK2 infection on cell response to drug and p53 activity.

Figure 14 presents induced p53 misfolding with inhibition of Ser46 phosphorylation in HIPK2 knockdown.

Figure 15 shows induced MT2A upregulation by knockdown of HIPK2.

Figure 16 exhibits the effect of Knockdown of MT2A in HIPK2 depleted cells on p53 transactivation activity.

Figure 17 presents p53 transcription activity in Zinc supplementation to HIPK2i cells.

Figure 18 shows changes in breast cancer tumors upon treatment with ADR and/or Zn, in vivo. Figure 19 exhibits up- and down-regulated genes in ADR/Zn treatment.

Figure 20 shows histological section of breast cancer tumors (in mice) 12 days after treatment with a combination of ADR and Zn, ADR alone or control (untreated).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides method for treating cancer, augmenting the tumor suppression activity of p53, augmenting the anticancer activity of an anticancer drug and reversing p53 dysfunction to p53 -induced oncosuppression.

The method of the invention is directed particularly to subjects suffering from cancer, wherein the cancer comprises cells expressing wild type p53.

The human p53 gene is mutated in about half of human cancers and in cancers harboring wild-type p53 (wtp53), its activity may be compromised by various mechanisms including deregulation of regulatory proteins. Various types of stress activate p53 mostly at post-translational level by a series of modifications, including phosphorylation and acetylation and the activated p53 leads to the expression of numerous target genes, including p21^Wafl and GADD45 for growth arrest, and PUMA, TNFRSF6/Fas-Apol, TNFRSF 10/Killer-DR5, and Noxa for apoptosis. Multiple molecular mechanisms contribute to p53 target gene selectivity and ample evidence indicates that the post-translational modifications of p53 may play a critical role in its target gene preference. It has been shown that p53 phosphorylation at serine 46 (Ser46) is a necessary step for inducing apoptosis in response to severe DNA damage. Moreover, defect in Ser46 phosphorylation contributes to the acquisition of the p53 resistance in an oral squamous carcinoma cell line. The transcriptional activation domain 2 (TAD2, residues 43-63) of p53 has been suggested to be necessary for mediation of apoptosis, because deletion of the TAD2 abolishes this activity and phosphorylation at both Ser46 and Thr55 enhances the binding to p53 of p62 and Tfbl, which play an important role in regulating p53 -target genes activation.

Several protein kinases have been involved in Ser46 phosphorylation, including p38, HIPK2, PKCδ, and DYRK (D'Orazi et ah, Nature Cell Biol. 4: 11-19, 2002). HIPK2 is a serine-threonine kinase able to regulate transcription. HIPK2 was shown to be activated by several types of DNA damage, including UV and ionising irradiation or treatment with cisplatin and roscovitine. In response to severe DNA damage HIPK2 specifically phosphorylates p53 at Ser46 regulating p53-induced apoptosis, by enhancing the p53 -mediated transcriptional activation of proapoptotic factors such as p53AIPl, PIG3, Bax, Noxa, and KILLER/DR5. Thus, the specific p53Ser46 phosphorylation is considered to be a sensor for DNA damage-intensity that promotes changes in p53 affinity for different promoters with a shift from cell-cycle-arrest-related genes to apoptosis-related genes. Of note, silencing of endogenous HIPK2 by RNA- interference induces tumor cell chemoresistance correlated with reduction of p53(p)Ser46 and abolishment of apoptosis. Reduced HIPK2 mRNA expression has been found in vivo in human breast, thyroid, and colon cancer tumors, suggesting an involvement for HIPK2 in restraining tumor progression (D'Orazi et al., Clin. Cancer Res. Hi 735-741, 2006). Furthermore, HIPK2 cytoplasmic relocalization, induced by HMGAl overexpression in breast cancers, inhibits p53 apoptotic function. Mutations of the HIPK2 gene have been found recently in acute myeloid leukemia and myelodisplastic syndrome, impairing p53 -mediate transcription activation. Recently, after the priority date of the present invention, it was suggested that different drug- activated pathways may regulate HIPK2 and that HIPK2/p53Ser46 deregulation is involved in chemoresistance (Puca et al., Gynecol Oncol., 2008 Jun; 109(3):403-10; Epub 2008 Apr l8).

As exemplified herein below, knock-down of HIPK2 in RKO colon cancer cells and MCF7 breast cancer cells resulted in the misfolding of p53 that acquired a "mutant- like" conformation, concomitant with the loss of its wild type transcriptional activity, particularly in response to adriamycine (ADR), and in the gain of enhanced tumor growth in vivo. It is further demonstrated that by combining ADR and zinc treatment the p53 regained its wild-type conformation, as detected by PAb240 and PAb 1620 antibodies, as well as the wild-type transcriptional activity. Additionally, the treatment of growing tumors in mice by the combination of ADR and zinc inhibited synergistically tumor growth significantly better than ADR alone. This represents a reversible switch between p53 tumor suppressor and tumor promoter which correlates with the conformational change as detected by the specific antibodies and points out to a possible way to affect the p53 activity in cancer, particularly in cases where wtp53 retains in the tumor. The wtp53 is intrinsically unstable and mutations usually reduce further its stability and inhibit the function of p53. Over 140 mutations were described in the DNA binding domain (DBD) that showed temperature- sensitive behavior where a small change of temperature can determine either loss or gain of tumorigenic function. According to the present invention, wtp53 may be affected by the lack of HIPK2 phosphorylation of Ser46 which is in the transcriptional activation domain (TAD), a portion of the intrinsically disordered N-terminal fragment of p53. Previous experiments demonstrated that this region, outside of the DNA binding domain (DBD), can also affect the DBD function since antibodies to this region, particularly pAblδOl that recognizes the epitope encompassed by residues 46-55, confers upon mutant p53 the regain of specific target DNA binding by the DBD.

HIPK2 plays an essential role in p53 regulation, after DNA damage, which is intimately linked to the activation of pro-apoptotic genes. Following ADR treatment HIPK2 phosphorylates p53 at Ser46, which is important for irreversible apoptotic commitment. Additional functions of HIPK2 independent from p53 regulation remain to be further characterized. The present invention discloses for the first time that HIPK2 knock-down contributes also to structural aspects of p53 and to its stability. The important role for Ser46 in p53 -induced apoptosis has been reported previously (Feng et al., Cell Cycle 5: 2812-2819, 2006), however, according to the present invention (p)Ser46 integrity may also be involved in p53 conformation as detected by the conformation specific antibodies (Figure 6), suggesting a pivotal role for this region of p53 in the control of the conformation of the entire molecule. The transcriptional activation domain 2 (TAD2, residues 43-63) of p53 has been shown to be necessary for mediation of apoptosis, since deletion of the TAD2 abolished this activity and phosphorylation at both Ser46 and Thr55 enhanced the binding to p53 of p62 and Tfbl, which play an important role in regulating p53 -target genes activation.

The present invention further discloses that HIPK2 depletion induces metallothioneins (MTs) overexpression in response to ADR treatment. MT is a class of cysteine-rich, metal binding anti-oxidant proteins that control the intracellular distribution of zinc and serve as a scavenger of reactive oxygen species. A number of studies have shown an increased expression of MTs in various human tumors. MTs also act as a potent chelator in removing zinc from p53 in vitro and may modulate p53 transcriptional activity. However, studies on the zinc dependent DBD folding suggest that DBD must fold in an environment where free Zn²⁺ concentration is low and carefully regulated by cellular metalloproteins and the actual free concentration of free Zn in the cell is not known. We therefore tried to rescue the misfolded inactive p53 in HIPK2i cells by adding zinc to the culture medium concomitant with ADR treatment. Surprisingly, zinc supplementation restored wtp53 transcription activity as evident by various assays (Figure 6). Phosphorylation of p53 in normal cells supplemented with zinc was recently shown (Shih et al., Exp. Biol. Med., 233:317-327, 2008). In addition, immunoprecipitation with conformation-specific antibodies showed a marked increase in p53 reactivity to PAbI 620 (folded/wild-type conformation). This observation together with the effect of antibody 1801 mentioned above suggest that a unique region within the N-terminus encompassing Ser46 may influences the folding of the DNA binding domain of p53. Moreover, by ChIP analysis, it was shown that the promoters occupancy by p53 shifted from the non canonical promoter (MT2A, "oncogenic" function) to canonical p53 target gene promoters (DR5, BTG2, p53R2, oncosuppressor function) following the ADR plus zinc treatment (Figure 6D). In conclusion, one can hypothesize that zinc may facilitate p53 binding to the target site in vivo, as it does in vitro, through regain of wild-type conformation state, although, the precise mechanism remains to be elucidated.

To evaluate this possibility the effect of ADR in combination with zinc was tested in vivo on tumor-bearing mice. Mice bearing tumors were treated with a combination of ADR and zinc. This treatment resulted in a significant synergistic inhibition of tumor growth (e.g. Figs. 6 and 18). Such effect on the HIPK2 -deficient tumors may be attributed to the regain of wtp53, as was shown in culture, and implies restoration of the tumor response to ADR treatment, although p53 -independent HIPK2 suppressor functions cannot be completely ruled out. The neoplastic potential due to reduction of HIPK2 function has been ascribed to impairment of p53 oncosuppressor functions. Here, our findings suggest a model in which loss of (p)Ser46 by HIPK2 leads to misfolded p53 state that allows some gain- of-function and switch from oncosuppressor to oncogene function. It is however significant that this "gain of function" attributed usually to mutant p53 is here exercised by wtp53. Thus wtp53 shows conformation dependent tumorigenicity.

The p53 is an intrinsically unstable protein belonging to the group of intrinsically disordered proteins and this property was particularly located to the N-terminal region encompassing Ser46. Intrinsically disordered proteins undergo disorder-to-order transition upon binding a target protein and may assume stable different conformation and even different activities upon binding of different targets. We show here that the wtp53 can acquire a mutant-like conformation likely depending on lack of Ser46 phosphorylation and that HIPK2i-derived tumors are more aggressive than control RKO or p53i-derived tumours. Since we found no difference between the tumour growth of H1299-HIPK2i and their control counterpart H1299 cell lines (p53 null), we assume that the increased aggressiveness of the HIPK2Ϊ tumors was due to the expression of wtp53 devoid of Ser46 phosphorylation. This novel finding, although seemingly counter-intuitive, correlates with recent results that showed growth promoting activity by p73, another tumor suppressor of the p53 family and with the finding that human p53 in the p53-null background of mice did not prevent accelerated tumor development after genotoxic or oncogenic stress, implying that wtp53 can lose its tumor suppressor activity. It also may correlate with the recent clinical analysis of ovarian cancer where cancer patients with wtp53 display a less favourable clinical response to chemotherapy and reduced long-term survival compared to those with mutant p53. The results on HIPK2 expression in colon cancer also support this contention and provide a possible mechanism to explain it, since low expression of HIPK2 was associated with poor outcome for patients with wtp53 tumors (p=0.05) but not in patients with mutant p53 (/?=0.45) (Figure 8). The possibility that wtp53 in vivo may fluctuate between growth promoter and growth suppressor depending on the cell cycle or stimulating factors was suggested in the past. Additionally, the question whether wtp53 that retained in tumors is a positive indicator for survival is still unanswered for many tumors.

In summary, the method of the invention induces at least one of the following clinical and physiological results in cancer cells, in vivo and in vitro:

(i) Regain of p53 transcription activity;

(ii) Restoration of response to anticancer drugs;

(iii) Induction of p53 to refold from an unfolded inactive confirmation to the folded reactive state; (iv) Reestablishment of p53 gene expression; and

(v) Augmentation of anticancer therapy. Thus, the method of the invention provides new clinical applications for affecting the gentle equilibrium between active and inactive wtp53 in many types of tumors towards increasing functional wtp53. Moreover, according to the present invention combination of chemotherapy and zinc inhibits enhanced tumor growth, in particular in cancers with wtp53.

The method comprises administering to a subject in need thereof a composition comprising zinc, with the proviso that the route of administration is other than topical administration. Alternatively, the method comprises administrating at least one anticancer agent in combination with the administration of zinc. Administering zinc or a pharmaceutically acceptable salt of zinc, in combination with at least one anticancer drug, according to the method of the invention, is via any suitable route, excluding topical administration of zinc over the skin at a specific location. The method of the invention is directed to a systemic treatment and thus it does not include topical application, such as, the application of ointments over the skin. Accordingly, the compositions of the invention may be administered by a variety of methods excluding topical administration. Thus, suitable routes of delivery include oral, parenteral, rectal, by inhalation or spray, in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes injection (bolus), infusion and the like. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Dosages of the disclosed compositions are similar to those already used in the art and known to the skilled artisan (see, for example, Physicians' Desk Reference, 54th Ed., Medical Economics Company, Montvale, N.J., 2000 hereby incorporated by reference in its entirety). However, the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.

The present invention provides a method for treating cancer. Hereinafter, the term "treatment" includes both the prevention of the genesis of cancer, as well as the substantial reduction or elimination of malignant cells and/or symptoms associated with the development and metastasis of malignancies. Cancers for which the therapeutic agents of the present invention are useful include, but are not limited to, breast cancers such as infiltrating duct carcinoma of the breast or other metastatic breast cancers, lung cancers such as small cell lung carcinoma, bone cancers, bladder cancers such as bladder carcinoma, rhabdomyosarcoma, angiosarcoma, adenocarcinoma of the colon, prostate or pancreas, or other metastatic prostate or colon cancers, squamous cell carcinoma of the cervix, ovarian cancer, malignant fibrous histiocytoma, skin cancers such as malignant melanoma, lymphomas and leukemia, leiomyosarcoma, astrocytoma, glioma and heptocellular carcinoma.

According to some embodiments, the method of the invention further comprises administering an anticancer drug in combination with zinc. The anticancer drug may be selected from the group consisting of: DNA damaging agents, DNA synthesis inhibitors, mitosis inhibitors, cell division inhibitors, antiangiogenic agents, hormonal derivatives, alkylating agents, antimetabolites, anti-proliferative agents, plant alkaloids, topoisomerase inhibitors or antitumour agents. As used herein, administration "in combination with" one or more anticancer agents includes simultaneous (concurrent) and consecutive administration in any order. According to the methods of the invention, the composition comprising zinc may be administered prior to, concurrent with, or following the anti-cancer compounds. The administration schedule may involve administering the different compounds in an alternating fashion and/or in different administration regimes. In other embodiments, the anticancer agent may be delivered before and during, or during and after, or before and after treatment with zinc, hi some cases, more than one anticancer therapy may be administered to a subject. For example, the subject may receive zinc, particularly zinc hydrochloride, in combination with both surgery and at least one anticancer compound.

Alternatively, zinc may be administered in combination with more than one anti-cancer drug. Examples of suitable anti-cancer drugs to be used in combination with the composition comprising zinc are well known and include: 3,2',3',4'- tetrahydroxychalcone; Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;

Adozelesin; Adriamycine; Aldesleukin; Altretamine; Ambomycin; Ametantrone

Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase;

Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate;

Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide;

Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin;

Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;

Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Depsipeptide; Dexormaplatin; Dezaguanine; Dezaguanine

Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;

Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin;

Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate;

Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide

Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; FK228;

Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin

Sodium; Gemcitabine; Gemcitabine Hydrochloride; Grape Seed Proanthocyanidin

Extract (GSPE); Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; IH636; Ilmofosine; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;

Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine;

Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine

Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril;

Mercaptopurine;. Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper;

Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole;

Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;

Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;. Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sirtinol; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin;. Streptozocin; Sulofenur; Talisomycin; Taxol; Taxotere; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Tenyposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Other compounds which are useful in combination therapy for the purpose of the invention include the antiproliferation compound, Piritrexim Isethionate; the antiprostatic hypertrophy compound, Sitogluside; the benign prostatic hyperplasia therapy compound, Tamsulosin Hydrochloride; the prostate growth inhibitor, Pentomone; radioactive compounds such as Fibrinogen I¹²⁵ and Fludeoxyglucose F¹⁸. Other compounds useful in combination therapies with the inhibitor compounds of the invention include anti-angiogenic compounds such as angiostatin, endostatin, fumagillin, non-glucocorticoid steroids and heparin or heparin fragments and antibodies to one or more angiogenic peptides such as alphaFGF, betaFGF, VEGF, IL-8 and GM- CSF. These aforementioned anticancer agents are administered along with zinc for the purpose of inhibiting proliferation or inhibiting tumor formation or metastasis formation in all of the conditions described herein. EXAMPLES Experimental Procedures

(i) Cell culture and treatments

The engineered C-RKO and HIPK2i colon cancer cells were generated by stable transduction of pSUPER vectors, as previously described (Di Stefano et al., Exp Cell

Res., 293: 311-320, 2004) resulting in the RKO-p53i, H1299-pSuper and H1299-

HIPK2i cell lines. Cells were cultured in RPMI-1640 (GIBCO-BRL) supplemented with

10% heat-inactivated fetal bovine serum, antibiotics, and 2.5 μM Hepes. For treatments,

ADR and ZnCl₂ were added to the culture medium to a final concentration of 2 μg/ml and 100 μM, respectively.

(ii) Hybridization and Analysis of Gene Expression by Microarrays

Total RNA from 0, 8, and 16 hr of culture with ADR was used to synthesize biotin- labeled cRNA and hybridized to separate Affymetrix HuI 33 A oligonucleotide arrays containing 22,215 probe sets (PS) (Affymetrix, Santa Clara, CA, USA). Gene expression values below 20 were adjusted to 20 to eliminate noise from the data, and subsequently all values were Iog2-transformed. Expression-level ratios for each group were determined at time points 8 hr and 16 hr, with respect to time 0 of the particular group. Only genes with expression ratios above 2 (or below 0.5) in one or both time points of a particular treatment group were selected. The modulated genes lists were presented in Venn diagrams that compared the common and specific genes in each treatment group. We applied the Sorting Points Into Neighborhoods (SPIN) algorithm on the up-regulated genes - an unsupervised method for analysis, organization, and visualization of the data.

Hybridization procedures and low-level processing were used for the human colon cancer samples. Survival plots were analyzed using the Kaplan-Meier (KM) method; survival of colon cancer patients with high (top) and low (bottom) expression levels of

HIPK2 (obtained from microarray gene expression data) were compared, separately for patients with wt and mutant p53.

(iii) RNA extraction, and RT-PCR

Cells were harvested in TRIzol Reagent (Invitrogen) and total RNA was isolated following the manufacturer's instructions. The first strand cDNA was synthesized according to the manufacturer's instructions (Moloney murine leukemia virus reverse transcriptase kit, Applied). Semi-quantitative Reverse-Transcribed PCR was carried out by using HOT-MASTER Taq (Eppendorf) using 2 μl cDNA reaction and genes specific oligonucleotides (Fig. 11) under conditions of linear amplification. PCR was performed in duplicate in two different sets of cDNA. PCR products were run on a 2% agarose gel and visualized with ethidium bromide. The housekeeping aldolase A and GAPDH mRNA, used as internal standard, were amplified from the same cDNA reaction mixture.

(iv) Chromatin Immunoprecipitation (ChIP) assay

ChIP analysis was carried out essentially as described (Di Stefano et al., Oncogene, 24: 5431-5442, 2005). Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were incubated overnight at 4⁰C with milk shaking using polyclonal anti-p53 antibody

(FL393, Santa Cruz Biotechnology). Before use, protein G (Pierce) was blocked with 1 μg/μl sheared herring sperm DNA and 1 μg/μl BSA for 3 hr at 4⁰C and then incubated with chromatin and antibody for 2 hr at 4⁰C. PRC was performed with HOT-MASTER

Taq (Eppendorf) using 2 μl of immuniprecipitated DNA and promoter-specific primers spanning p53 binding sites (Fig. 12). Immunoprecipitation with non-specific immunoglobulins (IgG, Santa Cruz Biotechnology) was performed as negative controls. PCR products were run on a 2% agarose gel and visualized with ethidium bromide.

(v) Viability, Clonogenic, and TUNEL assays

Exponentially proliferating cells were exposed to ADR (2 μg/ml) for different time points. Cells were counted in hemocytometer after addition of trypan blue. The percentage of viable cells, i.e. blue/total cells, was determined by scoring 100 cells per chamber for three times. For colony-formation assay, cells were exposed to ADR (2 μg/ml) for a pulse of 2 hr before replacing cell culture medium with fresh medium. ADR-induced death-resistant colonies were stained with crystal violet one week later. For Tunel assay, cells were treated with ADR for 24 hr, and subsequently fixed in 4% paraformaldehyde for 30 min at room temperature. After rinsing with PBS the samples were permeabilized in 0.1% Triton X-100 in sodium citrate, washed with PBS, and then incubated in the Tunel reaction mix for 1 hr at 37⁰C, according to the manufacturer's instructions (Roche, Germany). Cells were counter-stained with Hoechst before analysis with a fluorescent microscope (Zeiss).

(vi) Subcellular fractionation and Western blotting Whole cell lysates or cytoplsmic/nuclear fractions were resolved by SDS- polyacrilamide gel electrophoresis, as described (Di Stefano et al., 2004; ibid). Proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, Bedford, MA) and incubated with the first antibody followed by an anti-immunoglobulin-G- horseradish peroxidase antibody (BioRad). Specific proteins were detected by enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL). Antibodies against PARP and caspases-3 (both from BD Pharmingen), Ser46, (Cell Signalling), and p21 (C- 19, Santa Cruz), were used in accordance with the manufacturer's instruction. Tubulin (Immunological Sciences) and actin (SIGMA) were blotted for loading control.

( vii) Immunoprecipitation of p53 Cells treated with ADR (2 μg/ml) and ZnCl₂ (100 μM) for 24 hr were lysed in immunoprecipitation buffer (10 mM Tris, pH 7.6; 140 mM NaCl; 0.5% NP40, and protease inhibitors) for 20 min on ice, and cleared by centrifugation. Pre-cleared supernatants (400 μg) were immunoprecipitated overnight at 4⁰C with the conformation-specific monoclonal antibodies Pabl620 (wild-type specific, Calbiochem) and PAb240 (mutant specific, Oncogene Science, Inc.) pre-adsorbed to protein G- agarose (Pierce). Immunocomplexes were collected by centrifugation, separated by 9% SDS-PAGE and blotted onto PVDF membrane (Millipore). Immunoblotting was performed with anti-p53 PablδOl (Santa Cruz Biotechnology) antibody and the ECL chemoluminescence kit (Amersham). (viii) Transactivation assay

Cells were transiently transfected with the luciferase reporter gene driven by the p53- dependent promoters PG13-luc, Noxa-luc, and AIPl-luc using LipofectaminePlus (Invitrogen) method according to the manufacturer's instructions. Twenty-four hours after transfection cells were treated with ADR (2 μg/ml) for additional 12 hr. Transfection efficiency was normalized with the use of a co-transfected β-galactosidase plasmid. Luciferase activity was assayed on whole cell extract, as previously described (D'Orazi et al., 2002; ibid). The luciferase values were normalized to β-galactosidase activity and protein content.

(ix) Tumorigenicity in nude mice

Six- week-old CD-I nude (nu/nu) mice (Charles River Laboratories, Calco, Italy) were used for in vivo studies, as previously described (D'Orazi et al., 2006; ibid). Briefly, each experimental group included ten animals. Solid tumors were obtained by injecting

3x10⁶ viable C-RKO, HIPK2i, and p53i cells suspended in 0.2 ml PBS into the mice right-flank muscle (Int.). Their dimensions were measured every other day and their volumes were calculated from caliper measurements of two orthogonal diameters (JC and y, larger and smaller diameters, respectively) by using the formula VoIuHIe=Xy²/^.

For tumor treatments, HIPK2i cells were implanted Lm. on the flank of each mouse, allowing the tumors to grow to 400 mm³ weight (approximately 5-7 days from injection). Mice were then randomized in four groups (6-8 mice/group) and treated with ADR (10 mg/kg body weight), ZnCl₂ [10 mg (154 μmol) zinc/kg body weight intragastric], combination of ADR plus ZnCl₂, or PBS. ADR was injected once at day 7, Lp., while ZnCl₂ was administrated once daily, starting from day 7, over the course of two weeks. Tumor dimensions were measured every other day, as above. The antitumor effect of the combination treatment, ADR plus zinc, was evaluated by comparing the relative tumor size with tumors treated with ADR only or zinc only. All mouse procedures were carried out in accordance with Institutional standard guidelines.

(x) Statistical analyses

All experiment results were expressed as the arithmetic mean and standard deviation (s.d.) of measurements. Student's t-test was used for statistical significance of the differences between treatment groups in tumor growth. Statistical analysis was performed using analysis of variance at 5% (p<0.05) or 1% (p<0.01). Standard Kaplan- Meier analysis was performed.

(xi) Histological analysis

Explanted C-RKO, HIPK2i, and p53i xenografts were frozen in liquid nitrogen, paraffin embedded, sectioned, and stained with hematoxylin and eosin. Histological examination was carried out on transverse sections, 5 μm thick, of the whole cellular mass. From each sample 3 random selected sections were used for the morphometrical analysis. They were examined under a light microscope (Leica DMR, Leica Microsystems, Wetzlar, Germany) at a primary magnification of x 40 and the images of 10 randomly chosen fields per section were recorded using a digital camera (DC200, Leica Microsystems, Wetzlar, Germany). The nucleus/cytoplasm ratio was measured by computer-assisted image analysis using the QWin image analysis software (Leica Microsystems, Cambridge, UK). The analysis procedure involved a discrimination step based on a colour deconvolution procedure, used to separate haematoxylin- from eosin- stained structures, and followed by the application of the k-means thresholding algorithm to refine the identification of nuclear profiles. The nucleus/cytoplasm ratio was then estimated as the ratio between the total area covered by nuclear profiles and the total area of the cytoplasm. Data obtained from each specimen were averaged to provide a representative value for that specimen. Statistical comparisons between the experimental groups were tested by One-way Analysis of variance (ANOVA), followed by Bonferroni's test for multiple comparisons. p<0.05 was always considered as the limit for statistical significance.

Example 1- Knock-down of HIPK2 inhibits p53-dependent apoptosis and gene expression

It previously demonstrated that HIPK2 specifically phosphorylates p53 at Ser46 in response to severe DNA damage (D'Orazi et al., 2002; ibid), thus regulating p53- induced apoptosis. Here we compare the response of colon cancer RKO cells depleted of HIPK2 (HIPK2i) or p53 (p53i) to treatment with ADR. Cells depleted of HIPK2 and p53 (Figure IA) showed resistance to apoptotic cell death in response to ADR compared to control RKO cells (C-RKO), as assessed by cell death (Figure IB; Tripan blue exclusion, n=3), Tunel assays (Figure 1C and Figure ID - quantitation of apoptotic cells in (C) with respect to total nuclei stained with Hoechst), PARP and caspase-3 cleavages (Figure IE). C-RKO cells showed Ser46 phosphorylation while HIPK2i cells expressed p53 lacking of (p)Ser46 and p53i cells showed absence of p53 (Figure IE).

The colony assay showed complete rescue of the colony-forming efficiency, after ADR treatment, only in HIPK2Ϊ cells, while colony forming ability was strongly abolished in

C-RKO cells and only reduced in p53i cells (Figure IF). These findings suggest that, although both HIPK2i and p53i cells showed resistance to ADR-induced apoptosis, p53i cells still maintained drug response probably due to residual low level of wtp53 undetected by western blot.

To unravel the effect of HIPK2 knock-down and lack of p53Ser46 phosphorylation on gene expression we compared gene expression pattern of C-RKO, HIPK2i and p53i cells, following ADR treatment, by DNA microarray analyses. The expression data is threshold to 20 (i.e. all expression values smaller than 20 are set to 20), and Iog2 transformed. Expression ratios were determined for each group at time points 8 hr and 16 hr, with respect to the time 0 hr of each cell line. Only those genes that showed more than 2-fold induction or repression in one or both time points of a particular treatment group were considered and the genes that were modulated in the two time points were compared by the Venn diagram (Figure 2A). The modulated genes in C-RKO cells upon ADR treatment identified 1904 upregulated and 2791 down-regulated genes in at least one time point and among them were the known p53-targets involved in apoptosis, growth arrest, DNA damage, and stress response. The functional classification and expression data for selected genes upregulated by ADR treatment is presented in Figure 9 as genes activated in C-RKO cells. The genes presented in this figure correspond to the known p53 targets. These p53 target genes were not modulated in HIPK2i cells whereas some of them were still induced in p53i cells, including CCNG2/Cyclin G2, ATF3, and CDKNl A/p21 (Figure 9). The SPIN algorithm was used on the combined upregulated genes above two fold in both time points in all three cell lines as they appear in the Venn diagram (Fig. 2A), yielding 747 genes. This expression matrix (Figure 2B) confirmed that there was almost no overlap between genes upregulated in C-RKO and the interfered cells. The modulated genes in C-RKO in both time points were absent in HIPK2i and only few of them were present in p53i cells (Figure 2B). Analysis of 85 genes upregulated in HIPK2i showed that they were not induced in CRKO and p53i cells (Figure 2C); similarly, analysis of 226 genes upregulated in p53i cells showed that they were not induced in HIPK2i, however few of them were induced in C-RKO cells (Figure 2D). The microarray data were validated by reverse- transcriptase PCR analyses (RT-PCR) on a pool of activated genes, with an aliquot of the RNA used in the DNA chip analyses. The transcripts of the p53 target genes BTG2, NDRGl, Noxa, PIG3, and KILLER/DR5 were specifically induced only in C-RKO cells whereas the p21^Wafl transcript was also induced in p53i cells, in agreement with the microarray analysis (Figure 11). Figure 11 shows PCR of specific p53 target genes C-RKO, HIPK2i, and p53i cells collected at specific time points (0, 8, and 16 hr) of ADR (2 μg/ml) treatment for the microarray analysis. Data was validated by semiquantitative RT-PCR. Total RNAs was reverse-transcribed for PCR analyses. The mRNA levels were normalized to GAPDH and aldolase expressions. Total RNAs are also shown as control.

Consistent with this finding, chromatin immunoprecipitation (ChIP) analyses showed that p53 was bound to the selected target gene promoters in response to ADR only in C- RKO cells (Figure 3A) and not in HIPK2i cells (Figure 3B; *p<0.0l), and reporter luciferase assay showed that PG13-luc as well as Noxa- and AIPl-luc activities were induced by ADR treatment in C-RKO cells and significantly impaired by HIPK2 abrogation (Figure 3C). Overall, these data indicate that p53 target gene transcription in response to ADR is compromised by knock-down of HIPK2 to a larger extent than that of direct inhibition of p53 translation in p53i cells suggesting that p53 lost the wild-type functions upon depletion of HIPK2. Analysis of p53 subcellular distribution and phosphorylation does not show differences between C-RKO and HIPK2i cells (Figure 4A-B). An immunoblot blot analysis of p53 protein levels in nuclear (N) and cytoplasmic (C) fractions of C-RKO and HIPK2i cells treated with ADR is shown in Figure 4A. The separation of the cytoplasmic and nuclear fractions was confirmed by anti-tubulin and anti-histone-2A (H2A) antibodies, respectively (* - a non-specific signal). Figure 4B presents an immunoblot analysis of phosphorylation of p53 at Serl5 and Ser392, in C-RKO and HIPK2i cells treated with ADR.

Example 2- HlPK2i cells show marked enhancement of in vivo tumor growth In attempt to understand the consequence of p53 dysfunction in HIPK2i cells we compared the in vivo tumor growth of C-RKO, HIPK2i, and p53i cells. Surprisingly, HIPK2i cells exhibited significant enhanced tumorigenicity - tumor take was 100% for HIPK2i-derived tumors and 70 and 60% for C-RKO and p53i-derived tumors, respectively. HIPK2i-derived tumors exhibited a substantially shorter latency and enhanced growth compared to control and p53i-derived tumors and the median time of tumor appearance was 7, 18 and 24 days for HIPK2i, C-RKO, and p53i cells, respectively (Figure 5A; *p<0.05 vs C-RKO, ^*p<0.05 vs. p53i). Computer-image analysis of tumor sections (Figure 5B) showed a statistically significant increase of the nucleus/cytoplasm ratio (Figure 5C), usually associated by traditional histopathological criteria with a more malignant phenotype, in HIPK2i-derived tumors. To test whether the aggressiveness of these tumors was due to the deficiency in HIPK2 or to the dysfunctional wtp53 we compared H1299 cells (p53 null) with its counterpart H1299- HIPK2-interfered for the formation of tumors in nude mice under the same conditions as was done for the RKO cell lines. We did not observe increased tumor growth of H1299-HIPK2i-derived tumors indicating that the increased aggressiveness of the RK0-HIPK2i tumors was very likely due to the presence of dysfunctional p53 that gained tumorigenic activity. These data, along with the microarray data, suggest that HIPK2 depletion led to enhanced tumor growth compared to parental C-RKO and to p53i probably due to the loss of p53 wild-type functions associated with gain of tumorigenic function. However, other factors involved in cell growth and apoptosis regulated by HIPK2 independently from p53 cannot be ruled out.

Example 3 - Regain of wild-type-p53 activity in HlPK2i by zinc supplementation

Examination of the gene expression analyses in HIPK2i upon ADR treatment showed a marked overexpression (5-20 folds) of metallothioneins (MTs) in HIPK2Ϊ cells compared to C-RKO and p53i cells (Figure 11 and Figure 10). MTs act as potent chelator in removing zinc from p53 in vitro and modulate p53 transcriptional activity

(Meplan et al., Oncogene 19: 5227-5236 2000)

We supplemented the tissue culture medium with ZnCl₂ and found that zinc supplementation restored the response of HIPK2i cells to ADR treatment, as shown by cell death assay (Figure 6A), while it did not affect the ADR response of C-RKO cells (Figure 6A). Biochemical analyses showed that ZnCl₂ supplementation to HIPK2i cells induced down modulation of p53 expression to levels that are in accordance with wild- type p53 behaviour, upregulation of p21, and slight Ser46 phosphorylation in response to ADR (Figure 6B). Without wishing to be bound by any theory or mechanism, these data may indicate that zinc supply plays a role in the regain of the wtp53 transcription activity (e.g. p21 up-regulation). Moreover, p53 protein structure becomes now accessible to other known Ser46 kinases, e.g. p38, PKCδ, and DYRK that until now were unable to phosphorylate p53Ser46 without zinc addition, probably due to a p53 conformation that was inaccessible.

To determine if the zinc-dependent effect was the consequence of conformational change in the protein, endogenous p53 was immunoprecipitated from HIPK2i cells using the conformation-specific monoclonal antibodies Pab240 and Pabl620 . Whereas in HIPK2i cells p53 was mainly in the unfolded conformation recognized by Pab240, zinc supplementation induced p53 to switch, in response to ADR, from the unfolded

Pab240-reactive state (denoted "mutant-conformation"; Figure 6C) to the folded Pabl620-reactive state (denoted "wt-conformation"; Figure 6C), supporting the hypothesis that zinc may play a role in the control of conformation of the misfolded p53 and therefore in DNA-binding competence.

To further investigate the regain of wtp53 activity by zinc supplementation, the in vivo promoter occupancy by p53 in HIPK2i cells was analysed. We observed strong enrichment of p53 binding to its wild-type target promoters in response to ADR with zinc supplementation (Figure 6D). In contrast, we found that p53 was bound to MT2A promoter in HIPK2i cells and that this binding was abolished after zinc supplementation (Figure 6D). These findings suggest that the dysfunctional p53 binds to non-canonical promoters and that zinc supplementation allowed shift of p53 recruitment onto wtp53- dependent promoters. Analysis of the mRNA levels of p53 target genes indicated that wtp53 -dependent gene expression was essentially re-established in HIPK2i cells after zinc supplementation (Figure 6E). Collectively the results described so far suggest that wtp53 in HIPK2i cells can behave either as a mutant that lost tumor suppression, or as wild-type depending on conformational parameters and this conformational switch is reversible.

Example 4 - Tumor growth of EQPK2i cells can be inhibited by zinc supply in combination with ADR

The effect of ADR in combination with zinc on tumor growth was evaluated by implanting RKO-HIPK2i cells Im. into nude mice, allowing the tumors to grow to 400 mm³ and treating tumors with ADR and ZnCl₂ separately or in combination, as detailed in Experimental Procedures. Tumors untreated or treated with ZnCl₂ over the course of two weeks increased in size in a comparable manner reaching a volume of approximately 4000 mm³ by day 21 (Figure 7A; P- values less than 0.05 (*) and less than 0.001 (**) were deemed statistically significant). ADR treated tumors although showed a slower growth, displayed an average tumor volume above 3000 mm³ by day 21 that was not statistically significant compared to untreated or zinc-treated tumors (p=0.\77) (Figure 7A). Surprisingly, tumors treated with combination of ADR and ZnCl₂ showed a statistically significant reduction of tumor growth that remained below 2000 mm³ by day 21 (ADR+zinc vs ADR ^=0.0121; ADR+zinc vs zinc /?=0.0002) (Figure 7A). These results were confirmed by measuring the weight of each tumor isolated at day 21 (Figure 7B). The experiment was repeated twice and similar results were obtained each time. Taken together, these results support the notion that HIPK2 knock-down enhances tumor growth; likely through p53 misfolding that can be reversed by zinc supplementation, leading to restoration of cell response to ADR and synergism between zinc and ADR in inhibiting tumor growth.

Example 5 - Expression of HEPK2 is associated with poor survival of colon cancer patients

Our results may be particularly relevant to the effect of wtp53, retained in about 50% of cancer. For such a tumor low levels of HIPK2 may be indicative of bad outcome. To address this question we used expression microarray data on more than 300 samples from colon cancer patients with known clinical records and p53 mutation status (known for a subset of the patients). We used carcinoma samples as well as isolated homogeneous metastases containing tumour cells only, with no clean margins, as indicated also by the micro-array analyses, , and looked for association between HIPK2 expression and survival using the Kaplan-Meier procedure. From 60 wtp53 tumors we extracted the high and low tertiles of HIPK2 expression levels (N=20 patients in each group, ratio of average expression levels = 2.05). There were 105 mutant p53 tumors and we compared two groups of N=35 in the high and low tertiles of expression (ratio = 2.02). The results (see Figure 8) demonstrate that low expression of HIPK2 is associated with poor outcome in patients with wtp53, but not in those with mutant p53. These findings demonstrate that our results are relevant to human cancer. Example 6 - Effect of lentiviral LV-THsi/HIPK2 infection on cell response to drug and p53 activity

The constitutive HIPK2 depletion was first obtained by infecting MCF7 cells with the LV-THsi/HIPK2 lentiviral vector selected in view of the efficacy in transducing a broad range of human cancer cells. A non-specific LV-THsi vector was used as control (siRNA C). MCF7 cells were infected with LV-THsi/HIPK2 (HIPK2i) and the nonspecific RNAi lentiviral vectors (siRNA C; Figure 13A). One week after the infection HIPK2 mRNA expression was determined by reverse transcriptase-PCR (RT-PCR) analysis and normalized to GAPDH expression. Densitometric analysis of PCR products was performed and the HIPK2/GAPDH ratio was 1.9 and 0.7 for siRNA and HIPK2i, respectively. As shown in Fig. IA, the effectiveness of HIPK2 depletion (HIPK2i) is about 65% reduction).

Next, the cell response to drug treatment was assessed by viability assay in control and HIPK2i cells treated with ADR for 24 h. MCF7 were infected as detailed above and 1 week later MCF7/HIPK2i and siRNA C cells were treated with 0.5, 1, 1.5, and 2 μg/ml of ADR. Cell viability was assessed by trypan blue exclusion 24 h later (Figure 13 B; the results shown are representative of three independent experiments performed in duplicate. *p < 0.001). The results indicate that HIPK2 depletion results in significant resistance to ADR-induced cell death, whereas non-specific siRNA C cells shows major cell death with increasing amounts of ADR.

The involvement of p53 activity in response to ADR treatment was then tested by luciferase assay. To this end, the LV-THsi/HIPK2i MCF7 cells and the control counterparts were transiently transfected with the p21-luc reporter plasmid and 24 h later treated with ADR. The luciferase activity was determined following normalization to β-gal activity (Fig. 13 C). Data are representative of three independent experiments performed in duplicate and the results are expressed as Relative Luciferase Units (RLU). As shown in Fig. 1C, the luciferase reporter activity was induced by ADR treatment in siRNA C cells, whereas it was significantly impaired by HIPK2 depletion, suggesting that the p53 transactivation function was compromised.

Next we checked the binding of p53 to its target DNA sequences by chromatin immunoprecipitation (ChIP) analysis. MCF7/HIPK2i and siRNA C cells were treated with ADR and subjected to chromatin immunoprecipitation (ChIP) using polyclonal anti-p53 antibody (Fig. 13 D). Immunoprecipitates from each sample were analyzed by PCR using specific primers for p21 and DR5 promoters. Data are presented as fold of enrichment of p53 binding \op21 and DR5 promoters in response to ADR, calculated as mean relative ratios of p53/Input. The in vivo ChIP assay showed that the p53 recruitment onto the target gene promoters p21 and DR5 was enhanced in response to ADR treatment in siRNA C cells, whereas it was hampered in HIPK2i cells. These data show that in HIPK2 knockdown the wtp53 protein was impaired in its DNA-binding and transcription activities.

Example 7 - HEPK2 knockdown induced p53 misfolding with inhibition of Ser46 phosphorylation

To evaluate the status of p53 in MCF7/HIPK2i cells, the conformation of p53 protein was studied with immunoprecipitation technique by using Pabl620 and Pab240 antibodies, associated with wild-type and mutant conformation, respectively. Equal amounts of total cell extracts from MCF7/HIPK2i and siRNA C cells were immunoprecipitated with conformation-specific Pabl620 (for wild-type, folded conformation) and Pab240 (for mutant, unfolded conformation) antibodies. Western immunoblotting was performed with polyclonal anti-p53 antibody (Fig. 14A). The results show that HIPK2 depletion after infection with LV-THsi/HIPK2 lentiviral vector reduced the Pabl620-reactive (folded) phenotype and increased the reactivity to the PAb240 (unfolded p53 form), suggesting that indeed HIPK2 is important in maintaining p53 wild-type conformation.

The p53 nuclear accumulation and posttranslational modifications after ADR treatment were also evaluated. MCF7/HIPK2i and siRNA control cells were subjected to nuclear (N) and cytoplasmic (C) fractionation in the presence or absence of ADR and analyzed by Western immunoblotting with monoclonal anti-p53 antibody (DOl). Anti- tubulin and anti-Hsp70 antibodies were used to detect the cytoplasmic and nuclear fractions, respectively (Fig. 14B). Total cell extracts from MCF7/HIPK2i and siRNA control cells were analyzed by Western immunoblotting with anti-Serl5, -Ser46, -

Ser392, and anti-p53 in the presence or absence of ADR (Figure 14C; anti-tubulin was used as protein loading control). p53 expression in both siRNA C and HIPK2i cells was not detected at basal level while, after ADR treatment, p53 accumulated in the nucleus of siRNA C cells and to a lesser extent also of HIPK2i cells; on the contrary, the cytoplasmic accumulation was comparable between the two cell lines. Also some p53 phospho-Serine residues, e.g. Serl5 and Ser392, were comparably phosphorylated in siRNA C and HIPK2i cells whereas Ser46 phosphorylation was severely impaired in HIPK2i cells (Fig. 14C). These data suggest that the p53 dysfunction did neither affect p53 subcellular distribution nor cytoplasm/nucleus translocation following DNA damage, even though reduced p53 accumulation was observed in HIPK2i cells. However, HIPK2 knockdown induced p53 misfolding with reduced Ser46 phosphorylation after ADR treatment that strongly compromised p53 recruitment onto target gene promoters as well as its transactivation function in response to DNA damage.

Example 8 - Knockdown of HIPK2 induced MT2A upregulation It has been proposed that metallothionein might act as regulators of p53 activity and folding. We have recently found by DNA microarray analyses stronger induction of metallothionein in HIPK2i cells compared to the control cells (Puca et al., Cancer Res. 68:3707—3714, 2008). In the attempt to uncover the molecular mechanisms responsible of p53 misfolding following HIPK2 depletion, we focused on the expression of MT2A by using both stable and inducible HIPK2 interference as described hereinabove. Co- infections in MCF7 cells with LV-THsi/HIPK2 (that inhibits HIPK2) and LV-tTR- KRAB (that reverts HIPK2 inhibition) lentiviruses to create the Dox-inducible HIPK2 interference cell line (MCF7indsi/HIPK2) was performed. The HIPK2 and MT2A expressions were monitored by RT-PCR and normalized to GAPDH expression. The LV-THsi/HIPK2 infection alone reduced HIPK2 expression by nearly 80% and the co- infection of LV-tTR-KRAB rescued efficiently the cells from HIPK2 depletion (Fig. 15A). Interestingly, we found, in a comparable but opposite way, that MT2A expression increased following HIPK2 depletion and subsequently reduced to nearly the basal levels by LV-tTR-KRAB co-infection (Fig. 15A). We further characterized the engineered MCF7indsi/HIPK2 to evaluate the efficacy of inducing shRNA expression by doxycyclin (Dox) treatment. MCF7indsi/HIPK2 were treated with 1 μg/ml Dox. Five days later cells were harvested and the efficacy of HIPK2 knockdown was evaluated by RT-PCR analysis (Fig. 15B). Engineered MCF7indsi/HIPK2 cells were treated with Dox for 5 days before analyzing HIPK2 and MT2A expressions by RT-PCR. The mRNA levels were normalized to GAPDH expression. As shown in Fig. 3B, Dox was able to induce depletion of HIPK2 that again correlated with MT2A RNA upregulation.

Finally, MT upregulation in HIPK2 depleted cells, compared to control cells, was supported by increased MT protein levels as analyzed by Western immunoblotting (Fig. 15C). Total cell extracts from MCF7/HIPK2i and siRNA control cells were analyzed by Western immunoblotting with anti-MT antibody. Anti-actin was used as protein loading control. Altogether, these findings suggest that HIPK2 negatively regulated MT2A gene expression and that HIPK2 depletion correlated with MT upregulation. HIPK2 belongs to a family of homeodomain transcription factors and its role as transcriptional regulator, often in complex with histone deacetylases (including HDACl) has been demonstrated for several different transcription factors regulating cell survival and apoptosis.

Therefore, we asked whether HIPK2 could be recruited on MT2A promoter and be involved in chromatin remodelling for MT promoter activation. To this end, the recruitment of HIPK2, histone-deacetylase 1 (HDACl), and acetylated-Histone 4 (ac- H4) were analyzed by ChIP assay in MCF7/HIPK2i cells and the control counterpart (Fig. 15D). MCF7/HIPK2i and siRNA C cells were subjected to chromatin immunoprecipitation (ChIP) using specific polyclonal anti-HIPK2, anti-HDACl, and anti-ac-H4 antibodies or non-specific Ig. Immunoprecipitates from each sample were analyzed by PCR using specific primers for MT2A promoter. A sample representing linear amplification of the total input chromatin (Input) was included as control. As shown by the ChIP analyses HIPK2 was recruited onto MT2A promoter along with HDACl in control cells (non-specific RNA interference) while the amount of HIPK2 on MT2A promoter was strongly reduced upon HIPK2 depletion; in agreement, HIPK2 depletion resulted in the loss of HDACl occupancy and increased occupancy of the ac- H4 on MT2A promoter, suggesting that HIPK2 plays a role in MT2A regulation at the transcriptional level.

This hypothesis was also tested by transactivation assay using the MT2-luc reporter that showed, in agreement with the ChIP assay, that HIPK2 depletion significantly induced MT2A-luciferase activity (Fig. 15E). MCF7/HIPK2i and siRNA control cells were co-transfected with MT2A-luc reporter and β-gal plasmids. Thirty-six hours after transfection luciferase activity was determined following normalization to β-gal activity. Data represent mean ± S. D. of three independent experiments performed in duplicate, p < 0.01. Regulation of MT genes, in particular MT2A, has been widely studied. The promoter of this gene is complex, consisting of multiple cis-elements, involved in basal and induced transcription, furthermore, heavy metal ions may regulate human metallothionein gene transcription. Here we show, for the first time, that HIPK2 was bound to the MT promoter acting in a co-repressor complex along with HDACl .

Example 9 - Knockdown of MT2A in HIPK2 depleted cells restored p53 transactivation activity

As elevated metallothionein levels correlate with chemoresistance, increased cell proliferation, reduced apoptosis, and inhibition of p53 activity we sought to investigate whether MT2A was involved in p53 deregulation in HIPK2i cells. To this end, MCF7/HIPK2i cells were transiently transfected with siRNA for MT2A or control siRNA and 24 h later transfected with a plasmid containing 13 copies of the p53- binding consensus sequence upstream of a luciferase reporter gene (PG13-luc reporter). As shown in Fig. 16A, the PG13-luc was not induced in siRNA (Fig. 16A; p < 0.001). The results show that the PG13-luc was not induced in siRNA control after ADR treatment whereas it was significantly induced after siRNA for MT2A depletion, indicating restoration of p53 transcription activity.

Thus, RT-PCR analysis of mRNA levels of p53 target genes showed that p21 was induced by MT2A depletion in MCF7/HIPK2i cells at basal level, indicating restoration of p53 transactivation function as shown also by BTG2 expression that was induced in response to ADR only in MT2A depleted cells (Fig. 16B).

Moreover, the conformation of p53 protein was evaluated in MCF7/HIPK2i cells interfered for MT2A function in the presence or absence of ADR treatment. MCF7/HIPK2Ϊ cells were transfected with siRNA-control and siMT2A and 24 h later treated with ADR for 24 h. Equal amounts of total cell extracts were then immunoprecipitated with conformation-specific Pabl620 (for wild-type, folded conformation) and Pab240 (for mutant, unfolded conformation) antibodies. Western immunoblotting was performed with polyclonal anti-p53 antibody (Fig. 16C). The results indicate that MT2A depletion strongly increased the reactivity to the PAb 1620 (wild-type, folded p53 form); moreover, ADR treatment further increased the folded 1620-reactive conformer in siMT2A cells while strongly reducing the 240-reactive conformer, compared to the siRNA C cells (Fig. 16C). Therefore, MT2A interference could restore p53 transcription activity and wild-type conformation in HIPK2 depleted cells indicating that it was involved in p53 inhibition in HIPK2 knockdown. It has been suggested that MT can affect p53 activity, however, the role of MT in the control of p53 function is likely to be complex. Thus, it has been found that a small amount of MT can induce p53 activity by catalysing metal-transfer reactions regulating the folding of the DNA-binding domain; on the contrary, a large excess of MT reduces p53 transcriptional activity by exerting a metal chelator effect. Metal chelators can remove zinc from p53 turning the protein to a "mutant-like" form with the loss of the sequence-specific DNA- binding activity. Thus, metallothioneins control the intracellular distribution of zinc and also act as a potent chelator in abstracting zinc from p53 in vitro and may modulate p53 transcriptional activity.

Example 10 - Zinc supplementation to HIPK2i cells restored p53 transcription activity and drug-induced apoptosis In most cell types, zinc is often sequestrated through binding to metallothionein, keeping free zinc concentrations fairly low that could account for lack of function in a typical zinc-sensitive protein, including p53. To evaluate whether p53 targeted gene transcription was restored after zinc supplementation, RT-PCR analysis was performed in HIPK2i cells treated with ADR in the presence or absence of ZnCl₂ (Fig. 17A). Cells were treated with ZnCl₂ and ADR for 24 and 16 h, respectively before harvesting for RNA extraction. As shown in Fig. 17A, the mRNA levels were normalized to GAPDH expression. p21 was induced by zinc treatment, suggesting restoration of p53 transcription activity in HIPK2i cells.

To evaluate the biological outcome of zinc supplementation, MCF7/HIPK2i and control cells were treated with ADR in the presence or absence of zinc and 24 or 48 h later cell viability was assayed by trypan blue exclusion (Fig. 17B). The percentage (%) of cell death is shown by one representative experiment out of three performed in duplicate, p < 0.005. As shown in the figure, siRNA control cells underwent consistent cell death after ADR treatment, which did not significantly increase with zinc supplementation; on the contrary HIPK2 depletion strongly inhibited cell death in response to ADR that was restored only after zinc supplementation. Apoptotic cell death was evaluated by Western immunoblotting: MCF7/HIPK2i and siRNA C cells were treated with ZnCl₂ and ADR for 72 and 48 h, respectively, and the expression of PARP, Ser46 phosphorylation, and total p53 were determined by Western immunoblotting of nuclear cell extracts (Fig. 17C). The uncleaved (116 IcDa) and active cleaved (87 kDa) forms of PARP are shown by arrows. Hsp70 was used as protein loading control. The results show PARP cleavage and p53Ser46 phosphorylation in siRNA C cells treated with ADR and with combination of ADR and zinc. On the contrary, HIPK2i cells showed PARP cleavage only after combination treatment with ADR and zinc; interestingly, also p53Ser46 was restored likely by activation of residual HIPK2 or of other kinases known to phosphorylate Ser46 after ADR treatment (i.e., D YRK2).

The conformation of p53 protein was evaluated in MCF7/HIPK2i cells in the presence or absence of ADR and zinc treatments (Fig. 17D). MCF7/HIPK2i cells were treated with ZnCl₂ and ADR for 24 and 16 h, respectively. Equal amounts of total cell extracts were then immunoprecipitated with conformation-specific Pabl620 (for wild- type, folded conformation) and Pab240 (for mutant, unfolded conformation) antibodies. Western immunoblotting was performed with polyclonal anti-p53 antibody. As shown in Fig. 17D, the combination of ADR and zinc strongly reduced the Pab240 (unfolded) phenotype and increased the reactivity to the PAb 1620 (folded p53 form), compared to the single ADR treatment. These data suggest that zinc supplementation rescued p53 wild-type conformation and transcription activity and restored HIPK2-depleted cell response to ADR treatment. They also suggest that the fluxes of metal may control p53 activity.

Example 11 - Mouse model experiments: Breast cancer transgene (mammary adenocarcinoma), strain: FVBZN-Tg(MMTV neu)202Mul/J

Spontaneous breast tumors were allowed to grow to for one week after they were first palpated. Mice were then randomized in four groups (four to five mice per group) and treated with Adriamycin (10 mg/kg body weight), ZnCl₂ (10 mg zinc/kg body weight), combination of Adriamycin plus ZnCl₂, or PBS. Adriamycin was injected once at day 1, i.p., whereas ZnCl₂ was administrated once daily by oral administration, over the course of 12 days (Fig. 18A). Tumor dimensions were measured every other day and their volumes were calculated from caliper measurements of two orthogonal diameters (x and y, larger and smaller diameters, respectively) by using the formula volume = xy² / 2, as previously described. The antitumor effect of the combination treatment using zinc + Adriamycin was evaluated by comparing the relative tumor size with tumors treated with Adriamycin only or zinc only. All mouse procedures were carried out in accordance with institutional standard guidelines. The results indicate that treatment with ADR and Zn, but not with ADR alone, of mice bearing breast cancer and fed with regular nutrition, decreased tumor size. However, in mice fed with Zn-enriched nutrition, tumor increase was inhibited even with ADR alone (Fig. 18B) suggesting that Zn enrichment induced or enhanced the antitumor activity of ADR. In the absence of ADR treatment, tumor development was not affected by Zn-enriched nutrition (Fig. 18C). The combination of Zn/ ADR was effective in inhibiting tumor growth and reduced tumor size, in mice fed with regular or Zn-enriched nutrition (Fig. 18D). The results further indicated that antitumor effect of the Zn/ ADR combination was linearly correlated with tumor age (Fig. 18E). The younger the age of the breast cancer, the more efficient is the ADR+Zn treatment.

Example 12 - Mouse micro-array experiments

Methods: All experiments were performed using Affymetrix Mouse Gene l.Ost oligonucleotide arrays as described in http://www.affymetrix.com/support/technical/datasheets/gene_l_0_st_datasheet.pdf

Total RNA from each tumor sample was used to prepare biotinylated target DNA, according to manufacturers recommendations https://www.affvmetrix.com/support/downloads/manuals/wt sensetarget label manual, pdf. Briefly, 100-600 ng of total RNA was used to generate first-strand cDNA by using a T7-random hexamers primer. After second-strand synthesis, in vitro transcription was performed. The resulting cRNA was then used for a second cycle of first-strand cDNA by using a T7-random hexamers primer with UTP resulting in SS DNA used for fragmentation and terminal labeling.

The target cDNA generated from each sample was processed as per manufacturer's recommendation using an Affymetrix GeneChip Instrument System (^ttps://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual

.pdf). Specifically, spike controls were added to 5.5 μg fragmented cDNA before overnight hybridisation. Arrays were then washed and stained with streptavidin- phycoerythrin, before being scanned on an Affymetrix GeneChip scanner. A complete description of these procedures is available at (https://www.affvmetrix.com/support/downloads/manuals/wt_sensetarget label manual jxif).

Data analysis: Gene level RMA sketch algoritm (Affymetrix Expression Console and Partek Genomics Suite 6.2.) was used for crude data generation. Comparisons between samples were performed using various approaches and several different algorithms that were ran on the dataset. These included clustering, class prediction, statistical hypothesis testing (parametric or non parametric eg. t-Test, Mann-Withney), feature selection, principal components analysis and fold change calculations. Genes were filtered and analysed using unsupervised hierachical cluster analysis (Spotfire DecisionSite for Functional Genomics; Somerville,MA) to get a first assessment of the data. Further processing included functional analysis and over- representation calculations based on Gene Ontology and publication data: DAVID (http://appsl.niaid.nih.gov/David/upload.asp), Ingenuity, Database for Annotation (GO), Visualization, and Integrated Discovery. Over- representation calculations were done using Ease (DAVID).

Results: Figures 19A-C present of the number of genes up- and down-regulated in ADR/Zn treatment with respect to control. Figure 19A demonstrates the distribution between the up (>2 fold increase) and down (>2 fold decrease) regulated genes in the ADR+Zn treated compared with untreated tumors. The expression level of 534 genes was changed more than 2 fold upon ADR+Zn treatment in a distribution of 496 (93%) genes that were up-regulated and only 38 (7%) genes that were down regulated. The results are also presented with respect to the biofunction of the upregulated genes (Fig. 19B). The fact that vast majority of the genes were up-regulated upon ADR+Zn treatment indicates a strong stimulation of various pathways related to tumor cell death and tissue necrosis. This is exemplified by the decrease in tumor size and the necrotic tissue observed in breast cancer tumors following treatment with a combination of ADR and Zn (Fig. 20; lower panels). No change in tumor size or tissue was observed following treatment with ADR alone (Fig. 20; middle panels) or without any treatment ((Fig. 20; upper panels).. Indeed, the biofunction (Fig. 19B) clearly links the up- regulated genes to cellular pathways governing cancer, necrosis and cell death. These results at the molecular level reinforce the tumor measurement results and the pathology analyses performed on the same samples, indicating the strong and clear therapeutic effect of the ADR+Zn treatment in the breast cancer model mice.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method for treating cancer, comprising administering to a subject in need thereof a composition comprising zinc, with the proviso that the route of administration is other than a topical route of administration for the treatment of skin cancer and further comprising administering to the subject at least one anticancer drug.

2. The method of claim 1, wherein the composition comprises a pharmaceutically acceptable salt of zinc.

3. The method of claim 1, wherein the composition comprises zinc chloride.

4. The method of claim 1 , wherein the composition is administered via a route selected from a group consisting of: oral, parenteral, rectal, by inhalation.

5. The method of claim 4, wherein the composition is administered via a route selected from a group consisting of: intratumoral and intralesional, for other than skin cancers.

6. The method of claim 1, wherein the subject in need thereof is human.

7. The method of claim 1 , wherein the cancer comprises cancer cells expressing wild type p53.

8. The method of claim 7, wherein the cancer cells express low HIPK2.

9. The method of claim 1, wherein the cancer is selected from the group consisting of brain cancer, colon cancer, colorectal cancer, breast cancer, acute leukemia, lung cancer, kidney cancer, squamous cell cancer, testicular cancer, stomach cancer, melanoma, sarcomas, ovarian cancer, non-small cell lung cancer, esophageal cancer, pancreatic cancer, lymphoma, leukemia, neuroblastoma, mesothelioma, prostate cancer, bone cancer and hepatocellular cancer.

10. The method of claim 1, wherein the composition comprising zinc and the at least one anticancer drug are administered via different administration regimes.

11. The method of claim 10, wherein administering the at least one anticancer drug is selected from the group consisting of: administering the at least one anticancer drug prior to the administration of the composition comprising zinc, administering the at least one anticancer drug concurrent with the administration of the composition comprising zinc and administering the at least one anticancer drug following the administration of the composition comprising zinc.

12. The method of claim 11, wherein the anticancer drug is selected from the group consisting of: alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors or antitumour agents.

13. The method of claim 12, wherein the anticancer drug is adriamycine.

14. The method of claim 1, wherein the composition comprising zinc further comprises the at least one anticancer drug.

15. The method of claim 14, wherein the anticancer drug is selected from the group consisting of: alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors or antitumour agents.

16. The method of claim 15, wherein the anticancer drug is adriamycine.

17. The method of claim 1, wherein administering the composition comprising zinc and the at least one anticancer agent results in one or more therapeutic effects selected from the group consisting of: reduction in tumor size, induction of apoptosis in a tumor and inhibition of tumor cell proliferation.

18. Use of zinc or a pharmaceutically acceptable salt thereof and at least one anticancer drug for the preparation of a medicament for treating cancer, with the proviso that the medicament is other than a topical medicament for the treatment of skin cancer.

19. Use of zinc or a pharmaceutically acceptable salt thereof for the preparation of a medicament, with the proviso that the medicament is other than a topical medicament for the treatment of skin cancer, for treating cancer in combination with at least one anticancer drug.

20. The use according to any one of claims 18-19, wherein the medicament comprises a pharmaceutically acceptable salt of zinc.

21. The use according to any one of claims 18-19, wherein the medicament comprises zinc chloride.

22. The use according to any one of claims 18-19, wherein the cancer comprises cancer cells expressing wild type p53.

23. The use according to claim 22, wherein the cancer cells express low HIPK2.

24. The use according to any one of claims 18-19, wherein the cancer is selected from the group consisting of brain cancer, colon cancer, colorectal cancer, breast cancer, acute leukemia, lung cancer, kidney cancer, squamous cell cancer, testicular cancer, stomach cancer, melanoma, sarcomas, ovarian cancer, non-small cell lung cancer, esophageal cancer, pancreatic cancer, lymphoma, leukemia, neuroblastoma, mesothelioma, prostate cancer, bone cancer and hepatocellular cancer.

25. The use according to any one of claims 18-19, wherein the anticancer drug is selected from the group consisting of: alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors or antitumour agents.

26. The use according to claim 25, wherein the anticancer drug is adriamycine.

27. A composition comprising zinc and at least one anticancer drug for treating cancer, with the proviso that the composition is other than a topical composition for the treatment of skin cancer.

28. The composition of claim 27, wherein the anticancer drug is selected from the group consisting of: alkylating agents, antimetabolites, plant alkaloids, topoisomerase inhibitors or antitumour agents.

29. The composition of claim 28, wherein the anticancer drug is adriamycine.