Pathways of Dysregulation in Renal Cell Carcinoma: Rational Approaches to Development of Novel Treatment

Pathways of Dysregulation in Renal Cell Carcinoma: Rational Approaches to Development of Novel Treatment Mohamad K. Khasawneh Ronald M. Bukowski

Abstract Recent developments have involved a series of novel agents that produce clinical benefit in patients with advanced clear-cell renal cell carcinoma (RCC). The molecular characteristics of RCC, pathways involved in growth and progression, and development of targeted therapeutic approaches have become the focus of many investigators in the past decade. A variety of genetic abnormalities, molecular markers and drugs that target these markers or alter the genetic expression of certain regulatory proteins, have been identified and might have clinical significance for prognosis and treatment. However, specific markers associated with RCC and further development of novel single or combination targeted therapies is now required. An understanding of the complicated and unique biologic behavior of RCC and its various histologic subtypes is crucial for the continued development of novel and targeted therapies.

Cleveland Clinic, OH

Introduction Clinical Genitourinary Cancer, Vol. 5, Suppl. 1, S7-S18, 2006 Key words: Cytogenetics, Epidermal growth factor, Hypoxia-inducible factor, Mammalian target of rapamycin, Pathologic classification Submitted: Nov 17, 2006; Revised: Dec 12, 2006; Accepted: Dec 12, 2006 Address for correspondence: Ronald Bukowski, MD Cleveland Clinic 9500 Euclid Ave Cleveland, OH 44195-0001 Fax: 216-444-0114 E-mail: [email protected] Dr Khasawneh has no relevant relationships to disclose. Dr Bukowski has received research support from Bayer, Pfizer, Wyeth, Genentech, and Bristol-Myers Squibb. He is also a member of the Speaker’s Bureau for Pfizer and Bayer and has served as a paid consultant for or been on the Advisory Board of Bayer, Pfizer, Antigenics, Bristol-Myers Squibb, and Genentech. This article includes the discussion of investigational and/or unlabeled uses of drugs, including the use of bortezomib, lapatinib, and temsirolimus alone or in combination with interferon in metastatic renal cell carcinoma. It also includes discussion of axitinib, GW786034, vatalanib, and ARQ 197 in patients with metastatic renal cell carcinoma.

In 2006, approximately 39,000 people will be diagnosed and 13,000 will die from renal cell carcinoma (RCC) in the United States. Worldwide, the mortality from RCC exceeds 100,000 per year.1 Over the past 6 decades, there has been an increased incidence of RCC that has been attributed in part to the increased use of several abdominal imaging tools like ultrasound, computed tomography, and magnetic resonance imaging. In the United States, the incidence rates per 100,000 person-years increased by 2.3%, 3.1%, 3.9%, and 4.3% annually for white men, white women, black men, and black women, respectively.2 Risk factors for RCC include smoking, obesity, and hypertension, as well as acquired cystic kidney disease associated with end-stage renal disease. A 1.6:1 male predominance exists.3 Early detection and improved survival for patients with early-stage RCC has recently been reported.4 Despite this development, approximately one third of patients still present with advanced-stage and metastatic disease.5 Until recently, cytokines were the only therapy with meaningful benefit in limited subsets of patients with metastatic RCC.6,7 The response rates to interleukin (IL)–2 or interferon (IFN) alone were 10%-20% and usually were partial responses. Complete durable regressions associated with high-dose IL-2 were reported but involved highly selected patients and no overall survival (OS) benefit.8 The higher response rates and longer event-free survival obtained with IL-2/IFN must be balanced against the toxicity of such treatment.9 Patients who are refractory or intolerant to cytokine therapy provided significant challenges and represented an unmet medical need. The toxicity associated with cytokine therapy and the limited clinical benefit reported clearly indicated a need to develop new treatment approaches and strategies based on an understanding of RCC biology.

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Novel Therapeutic Approaches in Renal Cell Carcinoma

Pathologic Classification Six subtypes of epithelial renal tumors have been described based on morphologic, histochemical, electron-microscopic, as well as cytogenetic and molecular studies.10-12 The recognition that these histologic subtypes were not only different pathologically, but responded differently to therapy, was critical in development of novel therapy for this neoplasm.

Clear-Cell Carcinomas Clear-cell carcinomas comprise approximately 75%-85% of tumors and are characterized by a deletion of one or both copies of chromosome 3p.13 This is the most common variety and the group that responds to cytokine-based therapy.14

Chromophilic Carcinomas (Papillary) Chromophilic carcinomas (papillary) comprise approximately 14% of renal cancers, usually multifocal and bilateral, and present as small tumors (< 3 cm in diameter).15 Contrary to clear cell, these tumors do not contain 3p deletions16 but have monosomy Y, trisomy 7, and trisomy 17.17 This kind of tumor usually presents at a low stage and has a favorable outcome compared with clear-cell RCC. Clear-cell and chromophilic carcinomas originate from cells in the proximal tubule of the nephron18 and express cell-surface proteins normally found on such cells. The pattern of expression can further link the histogenesis of individual tumors to the proximal convoluted tubule or a proximal tubule progenitor cell.19

Chromophobic Carcinomas Chromophobic carcinomas comprise approximately 4% of RCCs that have a hypodiploid number of chromosomes and multiple chromosomal losses but no 3p loss.20 These patients are reported to have an excellent prognosis.21

Oncocytomas Oncocytomas are common tumors that are benign and do not metastasize.22

Collecting-Duct Carcinomas Collecting-duct carcinomas are extremely rare and clinically aggressive.23 In patients with advanced-stage disease, cisplatin-based chemotherapy can have activity, and as a group, these tumors behave more like transitional cell urothelial malignancies.24 No reproducible genetic alterations have been identified that are characteristic of oncocytomas or collecting-duct tumors.

The von Hippel–Lindau (VHL) tumor suppressor gene, which maps to chromosome subband 3p25, is mutated in hereditary RCC and in approximately 50% of sporadic clearcell RCC.25 Hereditary papillary renal carcinoma, which is distinct from the clear-cell variant associated with VHL disease, has been linked to germline gain of function mutations of the c-Met protooncogene or loss of function mutations of the Fumarate hydratase gene.26,27 Fumarate hydratase mutations are also associated with an increased risk of cutaneous leiomyomata and uterine fibroids.

The Von Hippel–Lindau Syndrome and Gene After the VHL gene was described in 1993, an improved understanding of RCC tumorigenesis and identification of potential biologic targets took place. Von Hippel–Lindau disease is an autosomal dominant cancer syndrome characterized by central nervous system and retinal hemangioblastomas, clear-cell renal carcinomas, and pheochromocytomas. The VHL gene consists of 3 exons and is widely expressed in fetal and adult tissues. Individuals with the VHL syndrome carry one wild-type VHL allele and one inactivated VHL allele, ie, VHL heterozygous. Tumor or cyst development in VHL disease is linked to somatic inactivation or loss of the remaining wild-type VHL allele. Biallelic VHL inactivation (secondary to mutation or hypermethylation) is seen in sporadic hemangioblastomas and sporadic clear-cell renal carcinomas.28 VHL somatic mutations occur in approximately 50%, and hypermethylation in another 15% of sporadic clear-cell renal carcinomas. However, mutation or hypermethylation of the VHL gene is rarely detected in other histologic subtypes of RCC. In sporadic tumors, the first “hit” and second “hit” occur somatically (that is, after conception) rather than in the germ line. The descriptions of the VHL syndrome and mutations of the VHL gene in the hereditary syndrome and sporadic clear-cell tumors provided the information and impetus for studies at the molecular and clinical level defining relevant therapeutic targets in this tumor.

Molecular and Biologic Targets in Clear-Cell Carcinoma: Hypoxia-Inducible Pathways

Unclassified Type

Clinical observations in patients with localized and advanced clear-cell tumors suggested that hypervascularity was a prominent feature of these neoplasms. The recent recognition of the importance of the VHL gene in development of this tumor, and the potential role of hypoxia-inducible factors (HIFs) in progression provided a clear rationale to investigate these pathways in this tumor.

Unclassified type is a poorly defined subgroup that can consist of several different variants.

Von Hippel–Lindau Protein

Cytogenetics Cancer-causing genetic alterations fall broadly into 2 functional classes: those that activate cellular genes, known as oncogenes, and those that inactivate cellular genes, known as tumor-suppressor genes.

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Von Hippel-Lindau messenger RNA (mRNA) encodes a protein (pVHL) that has 2 isoforms: pVHL19 and pVHL30, both of which appear to retain tumor suppressor activity. The term “pVHL” is used when referring to both isoforms. pVHL shuttles between the nucleus and cytoplasm.29-32 This shuttling by pVHL is important for its tumor suppressor function.

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Mohamad K. Khasawneh, Ronald M. Bukowski Figure 1 Von Hippel–Lindau Gene Product:

Figure 2 Vascular Endothelial Growth Factor

Normal and Aberrant Function A

Signaling Pathways

B pVHL

VEGF-A165 Vascular Tone Permeability Angiogenesis

pVHL Matrix

h.p. HIF-_

Ub Ub

Ub

Ubiquitin Attachment

NO

HIF-_ Constitutively Expressed HIF-_ Translocates into the Nucleus

PGs

KDR

Integrin _v ` 3

NICK VRAP Y951

HIF-_

Proteasome

HIF-_ Degradation

HIF`

Induction of Hypoxia-Inducible Genes, eg, VEGF, PDGF

SHP2 HCPTPA

Y996

NO

Src

?

eNOS

1054Y 1059Y

?

1175Y

Y1214

PLC-a

IP3

? PI3K

Migration Survival

pVHL30 resides primarily in the cytoplasm, whereas pVHL19 is located in the nucleus, suggesting that the functions of these 2 isoforms, although overlapping, are not identical.33 The tumors linked to VHL inactivation are often highly vascular and can overproduce angiogenic factors such as vascular endothelial growth factor (EGF; VEGF). In addition, RCC has been associated with erythropoietin overproduction.34 Vascular endothelial growth factor and erythropoietin mRNAs are prototypical hypoxia-inducible mRNAs, ie, these mRNAs are normally induced under conditions characterized by inadequate oxygenation. These considerations led to the discovery that cells lacking pVHL constitutively overproduce hypoxiainducible mRNAs and that restoration of pVHL function results in downregulation of hypoxia-inducible mRNAs in the presence of oxygen.35 Thus, overproduction of hypoxia-inducible mRNAs is a hallmark of pVHL-defective cells. Tissue microdissection, in situ hybridization, and immunohistochemical (IHC) studies of clear-cell RCC tumor specimens indicate that it is these cells, rather than the vascular components, that lack wild-type pVHL, which, in turn, overproduce HIF and its target gene products.36 Overexpression of HIF targets such as VEGF and platelet-derived growth factor–B (PDGF-B) are responsible for the hypervascularity of these lesions because VEGF and PDGF-B are potent mitogens for endothelial cells and pericytes, respectively. In addition, these stromal cells overproduce transforming growth factor–_ (TGF_) and its receptor, EGF receptor (EGFR), which suggests the establishment of an autocrine loop.37 Restoration of pVHL functions in VHL–/– renal carcinoma cells inhibits their growth in vivo. This indicates that biallelic VHL inactivation does not make renal carcinoma cells insensitive to pVHL function and suggests that “pVHL-mimetics” might be a therapeutic option for treatment.

PP2B

AKT

? Migration

PGs

PKC

Raf-1 eNOS

(A) In conditions of normoxia and normal VHL gene function, pVHL binds a hydroxylated proline residue of HIF-_, leading to ubiquitin attachment and degradation in the proteasome. (B) In hypoxia or with abnormal pVHL function, constitutively expressed HIF dimerizes with HIF-` and leads to transcription of hypoxia-inducible genes, including VEGF and PDGF. Abbreviation: h.p. = hydroxyproline

Ca2+

FAK

cPLA2

NFAT

COX2 Expression

MEK1/2 Bad ERK1/2

JNK

Survival Gene expression DNA Synthesis

Abbreviations: COX = cyclooxygenase; cPLA2 = cytosolic phospholipase A2; eNOS = endothelial constitutive nitric oxide synthase; ERK = extracellular signal–regulated kinase; FAK = focal adhesion kinase; HCPTPA = human cytoplasmic protein tyrosine phosphatase; IP3 = inositol 1,4,5-trisphosphate; JNK = c-Jun N-terminal protein kinase; KDR = kinase-insert domain-containing receptor; NFAT = nuclear factor of activated T cells; PI3K = phosphatidylinositol 3-kinase; PG = prostaglandins; PKC = protein kinase C; PLCa = phospholipase Ca; PP2B = protein phosphatase 2B; SHP2 = SH2 domain-containing protein tyrosine phosphatase; VRAP = VEGF-associated protein

Hypoxia-Inducible Factor Many genes regulated by hypoxia, like VEGF and Epo, are regulated by HIF. Hypoxia-inducible factor is made up of 2 nonidentical subunits (_ subunit and ` subunit) and binds to specific DNA sequences, which, in turn, activate transcription of many target genes that encode proteins involved in tumor angiogenesis, proliferation, differentiation, and other metabolic processes (Figure 1). These proteins include but are not limited to VEGF, PDGF receptor (PDGFR), TGF-_, EGF, glucose transporter-1, and erythropoietin.38,39

Vascular Endothelial Growth Factor Vascular endothelial growth factor is a dimeric glycoprotein and a member of the PDGF superfamily that includes VEGF-B, VEGF-C, VEGF-E, and placenta growth factor. Vascular endothelial growth factor exerts its biologic effect through interaction with transmembrane tyrosine kinase (TK) receptors present on the cell surface. These transmembrane TK receptors include VEGF receptor–1 (VEGFR-a; Flt-1) and VEGFR-2 (kinase insert domain-containing receptor/Flk-1), selectively expressed on vascular endothelial cells, VEGFR-3 (Flt-4), expressed on lymphatic and vascular endothelium and neurons.40,41 After VEGF binds to the extracellular domain of the receptor, dimerization and autophosphorylation of the intracellullar receptor TKs occurs and a cascade of downstream proteins are activated (Figure 2).42

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Novel Therapeutic Approaches in Renal Cell Carcinoma Vascular endothelial growth factor receptor-2 is the main receptor responsible for mediating the proangiogenic effects of VEGF. Vascular endothelial growth factor is the most potent described proangiogenic protein studied so far, especially in the setting of RCC. In RCC, VEGF expression results from inactivation of the VHL tumor-suppressor gene observed in the majority of RCCs.

this reason, it might be useful to combine a VEGF antagonist with an inhibitor of the HIF-responsive pericyte growth factor PDGF-B (also known as horizontal strategy). Vascular endothelial growth factor and PDGFRs share structural similarities, and for this reason, some small-molecule kinase domain-related inhibitors also inhibit the PDGFR. Examples of such molecules include sunitinib and sorafenib.

Platelet-Derived Growth Factor Platelet-derived growth factors are a family of proteins that exists in the A, B, C, or D forms. Platelet-derived growth factors are involved in proliferation pathways, especially of mesenchymal cell types. Platelet-derived growth factors form homodimers (eg, AA, BB, CC, or DD) or heterodimers (eg, AB), which interact with appropriate cellular receptors, which bind to 3 different TK receptors (PDGFR-_, PDGFR-_`, and PDGFR-``). Platelet-derived growth factor receptor–` is more frequently expressed in malignant cells. Platelet-derived growth factors induce DNA synthesis, growth, and inhibition of apoptosis in cells expressing PDGFRs.43 Platelet-derived growth factor–_ expression correlates with higher nuclear grade (grade 3/4) compared with lower grade tumors, and this expression is a predictor of tumor progression on univariate analysis and might be an unfavorable prognostic marker.37

Carbonic Anhydrase IX Carbonic anhydrase IX (CAIX) regulates cell membrane ion channels and extracellular pH, which might contribute to invasiveness and the metastatic behavior of cancer cells. Carbonic anhydrase IX is expressed in approximately 95% of clear-cell RCCs and not found in normal renal tissue. Carbonic anhydrase IX is regulated by the HIF-1 transcription factor.43 Recently, the level of CAIX expression defined by IHC using a specific monoclonal antibody (MoAb) was reported to predict response to high-dose IL-2 in patients with clear-cell carcinoma.44

Therapeutic Approaches Targeting Hypoxia-Inducible Pathways The biologic and clinical relevance of the VHL/VEGF pathways in clear-cell carcinoma resulted in a series of laboratory and clinical investigations to determine whether agents inhibiting or interrupting defined molecular structures and pathways produced clinical effects. A series of agents in a variety of categories have now been used clinically. Because many single agents have proven clinical benefit with tolerable toxicities, this made many researchers think about combining agents that block the HIF-VEGF pathway at multiple levels (also known as vertical strategy), for example, combining a compound such as rapamycin, which downregulates HIF levels through mammalian target of rapamycin (mTOR) inhibition, with agents that bind VEGF (such as bevacizumab) or small molecules that inhibit its receptor (such as PTK787). Preclinical studies suggest that established, mature blood vessels have a diminished requirement for VEGF for survival relative to newly sprouting vessels because of auxiliary survival signals provided by surrounding pericytes and stroma.45,46 For

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Vascular Endothelial Growth Factor Inhibitors Bevacizumab is a humanized MoAb against VEGF that was created from the murine A.4.6.1 antibody.47 It binds and neutralizes all major isoforms of VEGF-A. It was assessed in phase I trials as a single agent and in combination with chemotherapy,48,49 in randomized phase II/III trials in RCC to define its effects. A series of low molecular weight, orally bioavailable inhibitors of TKs involved in clear-cell carcinoma proliferation, growth, and progression has been developed. A brief description of this group of agents and the relevant targets inhibited are as follows. All of these might have significant effects in advanced-stage RCC. Bay 43-9006. Bay 43-9006 (sorafenib) is a multitargeted inhibitor with activity against several TKs such as those for VEGFR-2, VEGFR-3, PDGFR-`, p38, and c-Kit among others.50 Sorafenib inhibits the Raf/MEK/extracellular signal–regulated kinase pathway as well as phosphorylation of VEGFR and PDGFR. SU11248. SU11248 (sunitinib) is a novel agent that is a multitargeted receptor TK inhibitor exhibiting direct antitumor activity against tumor cells dependent on signaling through PDGF, KIT, and Flt-3 receptors for proliferation and survival. This is in addition to its inhibitory effects of EGFR-1 TK activity and its antiangiogenic activity through its potent inhibition of VEGFR-2 and PDGFR-` signaling.51 AG-013736 . AG-013736 (axitinib) is a selective inhibitor of VEGF and PDGF-` receptor TKs. A phase I trial was done in patients with different malignancies, and axitinib was found to be safe and acceptable; dose-limiting toxicities including hypertension, increased liver function tests, and stomatitis.52 GW786034. GW786034 is a small molecule that is a potent antiangiogenic agent through inhibition of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-` TKs, and c-Kit. A phase I trial showed antitumor and antiangiogenic activities of this agent.53 It is generally well tolerated, and common adverse events included nausea, diarrhea, fatigue, hypertension, and anorexia. PTK787/ZK222584. PTK787/ZK222584 is a selective inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, and PDGFR-` TKs, and was shown to inhibit VEGF and PDGF-induced tumor vascularization in preclinical models. It was used in various malignancies, and toxicities included fatigue, lightheadedness, nausea, and vomiting.54,55 Tumor blood supply was assessed using dynamic contrast-enhanced magnetic resonance imaging, and significant reductions in tumor blood supply were noted.

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Mohamad K. Khasawneh, Ronald M. Bukowski AZD 2171. AZD 2171 is a potent inhibitor of VEGF receptor TK that demonstrated an acceptable safety profile using an oral form in patients with advanced malignancies.56 Further assessment of this product in patients with RCC is planned in future clinical trials.

Figure 3 EGFR Signaling Pathway Enzyme Inside the Cell Membrane AEGFR TK: AnThat Regulates Growth

Hypoxia-Inducible Factor Inhibitors PS-341 or bortezomib is a dipeptide boronic acid derivative that inhibits the proteasome by stabilization of its active site and by reversible inhibition of its chymotrypsin-like activity. The ubiquitin-proteasome pathway plays an essential role in the degradation of many intracellular proteins, including HIF.57 It has been mentioned that HIF-1, another target of the ubiquitin-proteasome pathway, accumulated in the presence of proteasome inhibitor in a functionally inactive form. Two hypotheses were formulated; the first presumed that stabilization of the HIF-1 protein on its own is not sufficient to generate a functional form because additional regulatory steps are required to induce transcriptional activity of HIF-1.58 This theory correctly anticipated the regulation of HIF-1 transcriptional activity by hydroxylation of N803,59 but it does not provide an explanation for inactive HIF-1 in hypoxia. In the second theory, polyubiquitylation of HIF-1 interfered with the ability of HIF-1 to mediate hypoxic signal transduction and impeded nuclear transport.58 Several phase II trials of PS-341 in patients with metastatic RCC have been reported. Only limited clinical activity was noted in the presence of significant toxicity. Grade 3/4 toxicities included thrombocytopenia, arthralgias, anemia, febrile neutropenia, neuropathy, electrolyte disturbances, gastrointestinal, pain, and fatigue. Attempts to measure inhibition of proteasome activity with this agent were not successful. Additional inhibitors of HIF are in development but have not entered clinical trials in RCC as yet.60,61

Carbonic Anhydrase IX Inhibitors G250 is an immunoglobulin G MoAb that binds and inhibits CAIX. Infusions of G250 have been evaluated in combination with subcutaneous IFN-_2a62 or IL-263 in advanced-stage RCC. Toxicity was reported as acceptable, but no evidence of enhancement of the cytokine’s clinical activity was detectable.

In normal cells, the EGFR TK signal is strictly regulated. Therefore, cell growth is controlled.

Events That Can Turn on the EGFR TK Signal Outside the cell Ligand binding of EGFR Overexpression of EGFR

EGFR TK

B

Inside the cell Overexpression of EGFR Crosstalk with other receptors Loss of regulatory mechanisms EGFR mutations

Turning on the EGFR TK Signal: A Pivotal Event in Malignancy In tumor cells, the EGFR TK signal is inappropriately turned on EGFR TK drives uncontrolled cancer cell growth

EGFR TK

Epidermal Growth Factor/Epidermal Growth Factor Receptor Pathway Epidermal Growth Factor Receptor. The HER family consists of multiple transmembrane receptors; HER1/EGFR (ErbB-1), HER2 (ErbB-2 or neu), HER3 (ErbB-3), and HER4 (ErbB-4; Figure 3).64 The EGFR content was increased significantly in RCC compared with normal tissues. In all cases in which EGFR was undetectable, there was no evidence of distant metastasis, venous invasion, or regional lymph node involvement, and these patients had a better clinical outcome than patients with detectable EGFR. The EGFR content was significantly lower in nuclear grade 1 tumors than in tumors of higher nuclear grades. No significant difference between EGFR content and other clinicopathologic findings was

EGFR TK

Inhibition of Apoptosis

Proliferation

Invasion

Metastasis Angiogenesis

Epidermal growth factor receptor is a cell surface receptor that dimerizes on ligand activation of the extracellular binding domain. Dimerization leads to activation of a TK in the receptor’s cytoplasmic domain. Subsequent phosphorylation of the receptor’s intracellular tyrosine residues initiates signal transduction. Aberrant activation of the EGFR TK system in tumor cells derives from extracellular and intracellular events. The result is initiation of signal transduction programs associated with malignant progression.

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Novel Therapeutic Approaches in Renal Cell Carcinoma Figure 4 Phosphoinositide Signaling Pathway

PI3K P

P

PIP2 P

H O O OH H H H

P

P

PIP3 P

P

H O O OH H H

P

PTEN Growth and Survival Signal P

Phosphatidylinositol 3-kinase is a lipid kinase catalyzing transfer of a phosphate group to phosphatidylinositol-4,5 bisphosphate, generating phosphatidylinositol-3,4,5 triphosphate, which transmits growth and survival signals. Phosphatase and tensin homologue deleted on chromosome 10 removes D3 phosphate from phosphatidylinositol-3,4,5 triphosphate, inactivating the signaling cascade and regenerating phosphatidylinositol-4,5 bisphosphate.

detected.65 The demonstration of EGFR overexpression in RCC has provided the rationale for investigation of agents inhibiting this pathway. Activating mutations of EGFR have not been found in RCC, but it is possible that EGFR expression levels in RCC might be associated with outcome and/or prognosis in RCC. Transforming Growth Factor–_. Transforming growth factor receptor–_ activates the transmembrane TK receptor EGF HER1/ EGFR (ErbB-1). It is overproduced early in the course of RCC and believed to be secondary to HIF activation in VHL –/– RCC. It induces angiogenesis and cell proliferation.66 Tumor growth factor receptor–_ can also act as a specific growth-stimulatory factor for VHL –/– RCC cells expressing reintroduced wild-type VHL, as well as primary renal proximal tubule epithelial cells, the likely site of origin of clear-cell RCC.67 This role is in contrast to those of other growth factors overexpressed by VHL –/– RCC cells, such as VEGF and TGF-`1, which do not stimulate RCC cell proliferation. A TGF-_–specific antisense oligodeoxynucleotide blocked TGF-_ production in VHL –/– RCC cells, which led to the dependence of those cells on exogenous growth factors to sustain growth in culture.68 Growth of VHL –/– RCC cells was also significantly reduced by a panitumumab that specifically inhibits the EGFR, the receptor through which TGF-_ stimulates proliferation. These results suggest that the generation of a TGF-_ autocrine loop as a consequence of VHL inactivation in renal proximal tubule epithelial cells might provide the uncontrolled growth stimulus necessary for the initiation of tumorigenesis.62 The TGF-_ pathway is therefore a logical choice for therapeutic intervention.

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Transforming Growth Factor–`. There are at * 5 isoforms, and the prototype is TGF-`1, which is produced by proximal tubular cells and renal cancer cells.69 Tumor growth factor receptor–` is mainly an inhibitory growth factor with various functions like promoting epithelial cell differentiation, regulating interactions with extracellular matrix, in addition to inhibition of their proliferation.70 The various TGF-` isotypes share many biologic activities, and their actions on cells are qualitatively similar in most cases, although there are a few examples of distinct activities. In some systems, TGF-`3 appears to be more active than the other isotypes. Tumor growth factor receptor–`2 is the only variant that does not inhibit the growth of endothelial cells. Currently there are 2 TGF-` receptors (TBR1 and TBR2) involved in signal transduction.71

Therapeutic Approaches Targeting EGF/EGFR Pathways: EGFR Inhibitors Despite the demonstration of EGFR overexpression in RCC, clinical trials using a variety of EGFR inhibitors have not demonstrated consistent clinical benefit. Several of these agents are reviewed in this section.

Monoclonal Antibodies ABX-EGF. ABX-EGF or panitumumab is a fully human immunoglobulin G2 MoAb that specifically binds human EGFR and produces significant inhibition of tumors overexpressing EGFR in preclinical models.72 In a series of phase II trials, the antibody was well tolerated, however, it demonstrated only limited activity in patients with metastatic RCC. C225. C225 or cetuximab is an anti-EGFR chimerized MoAb. A phase II trial in patients with advanced-stage RCC did not demonstrate significant antitumor activity.73

Tyrosine Kinase Inhibitors OSI-774. OSI-774 or erlotinib is a selective inhibitor of HER1/EGFR TK that has in vitro and in vivo activity against various tumors.74 ZD1839. ZD1839 or gefitinib is a selective EGFR TK inhibitor that has direct inhibitory effects on cell proliferation and angiogenesis of renal carcinoma cell lines, however, without significant antitumor activity or improvement in progression-free survival in phase II trials in patients with metastatic RCC.75 GW572016. GW572016 or lapatinib is a dual inhibitor of type I TK for HER1/EGFR and HER2/ErbB-2. Overexpression of both receptors in certain malignancies is associated with more aggressive growth pattern and poorer prognosis.62,76 This agent binds to the cytoplasmic adenosine triphosphate–binding site of the kinase and prevents its phosphorylation. A randomized controlled phase III clinical trial (study EGF20001) in patients with advanced-stage or metastatic RCC that has not responded to first-line cytokine-based therapy was recently reported.77 Lapatinib was compared with

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Mohamad K. Khasawneh, Ronald M. Bukowski Figure 5 PI3K/AKT and mTOR Signaling Pathways A

B IGF-1

Nutrients ATP P

IGF-1 Receptor

Akt

P

PDK

P P P

P PI3K

TSC1 P P TSC2

P

Akt

PDK

P

P

TSC2

Cell Cycle Cell Survival Metabolism DNA Damage

mTOR

P

P P

P P P Forkhead

P

P

Bad

P

GSK 3

Cell Metabolism Survival Cell Cycle

mTOR Nutrient Response Cell and Organ Size Cell Cycle Protein Translation

p70s6 kinase

Rapamycin P

4EBP1 P

S6 Translation

(A) Binding of growth factor ligands activates kinase receptors, leading to recruitment of PI3K to receptor complex. Activated PI3K phosphorylates phosphatidylinositol-4,5 bisphosphate, which, in turn, activates phosphoinositide-dependent kinase–1 and Akt, leading to phosphorylation and inhibition of downstream substrates. (B) Mammalian target of rapamycin pathway can receive upstream inputs from the PI3K pathway and unknown sensors of nutrients, glucose, or energy. The PI3K signal can be transmitted through phosphorylation of TSC2 by Akt, leading to inhibition of TSC2 and then activation of mTOR, through Akt phosphorylation and activation of mTOR, or through phosphoinositide-dependent kinase–1 phosphorylation of p70s6 kinase. Arrows and T lines indicate activating and inactivating connections, respectively. Connections where mechanism is known are black and where mechanism is unknown gray. Abbreviations: ATP = adenosine triphosphate; 4EBP1 = eukaryotic initiation factor 4E-binding protein; GSK = glycogen synthase kinase; IGF = insulin-like growth factor; PDK = phosphoinositide-dependent kinase; PI3K = phosphatidylinositol 3-kinase; TSC = tuberous sclerosis

hormonal therapy in patients with advanced-stage RCC, and differences in overall survival or progression-free survival were seen. A subset analysis using patients with * 3 EGFR expression was then performed. When compared with hormonal therapy, lapatinib therapy significantly improved survival. Confirmation of this observation is awaited.

PTEN/PI3K/Akt Pathway In a study of 48 patients with RCC, a significant association of high Akt activation with metastasis and with tumor grade were noted. In addition, patients with high Akt activation had a significantly shorter cancer-specific survival than those with low activation, so an increased Akt activation could have an important role in the pathogenesis and progression of RCC. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that removes phosphate groups from key phosphoinositide-signaling molecules (Figure 4).78 This activity normally serves to restrict growth and survival signals by limiting activity of the phosphatidylinositol-3 kinase (PI3K) pathway. Tumor cells that lack PTEN suppressor gene lead to constitutive activation of downstream components of the PI3K pathway including the Akt and mTOR kinases. Nuclear signals for cell growth and survival are transmitted by receptor TKs. When receptor TKs are activated via ligand binding, PI3K is activated, which results in further recruitment of PI3K and the generation of phosphatidylinositol-4,5 bispho-

sphate (Figure 5). Phosphatidylinositol 3,4,5-trisphosphate, in turn, recruits kinases including the protein kinase B/Akt family of kinases and phosphoinositide-dependent kinase–1.79 After Akt is activated, it phosphorylates further targets (Figure 5) that have crucial functions in the regulation of proliferation, apoptosis, glucose homeostasis, cell size, nutrient response, and DNA damage.80 Inactivation of the PTEN suppressor genes in human cancer cell lines and in some animal models leads to activation of this pathway,81 which can be reversed by alterations in PI3K or Akt activity or by alterations in further downstream members of the pathway. In short, loss of PTEN results in cellular changes within the PI3K/Akt pathway to compensate for that loss. The mTOR pathway can receive upstream inputs from the PI3K pathway and unknown sensors of nutrients, glucose, or energy. The PI3K signal can be transmitted through phosphorylation of TSC2 by Akt, leading to inhibition of TSC2 and then activation of mTOR, through Akt phosphorylation and activation of mTOR, or through phosphoinositide-dependent kinase–1 phosphorylation of p70s6 kinase. This pathway regulates the cellular response to starvation or growth conditions such as amino acid deprivation, as well as controlling cell size, organ size, and proliferation. In animal models, it was demonstrated that mice with nonfunctional PTEN had an increased cell and organ size, whereas overexpression of d-PTEN yields the opposite phenotype.82,83

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Novel Therapeutic Approaches in Renal Cell Carcinoma

Therapeutic Approaches Targeting the PI3K/Akt/mTOR Pathway Inhibition of PI3K Currently, there are 2 experimental PI3K inhibitors available, wortmannin and LY294002. Both have demonstrated marked antitumor cell activity in vitro, particularly in PTEN-null cells or in cells overexpressing PI3K.81 Clinical trials in patients with RCC have not been reported.

Inhibition of Akt In mouse embryonic stem cells, inactivation of Akt1 significantly attenuates tumor formation resulting from loss of PTEN.84 In RCC tumors, the phosphorylation of Akt and its relationship to prognosis have been investigated. Akt activation was evaluated by IHC in 48 patients with RCC. Increased Akt was significantly associated with tumor grade and metastatic disease, whereas it was not associated with tumor stage or histologic subtype. In addition, it was significantly associated with a poor cancer-specific survival rate on univariate analysis. Several inhibitors of Akt are in clinical development including perifosine. Perifosine (octadecyl-[1,1-dimethylpiperidino-4-yl] phosphate; D-21266; NSC639966) is an analogue of miltefosine with greater oral bioavailability currently undergoing clinical evaluation.85 Alkylphospholipids are known to alter several aspects of cell membrane synthesis and function, including inhibition of phospholipase C, guanine nucleotide-binding protein, protein kinase C activity, and phosphatidylcholine synthesis.86 Perifosine has demonstrated rapid decrease in the phosphorylation of Akt (PKB), with loss of Akt activity87,88 and decreased translocation of Akt to the plasma membrane.

Inhibitors of Mammalian Target of Rapamycin Rapamycin is a natural antibiotic that inhibits the mTOR pathway. As a result, rapamycin has been used as an immunosuppressant in transplantation patients because it blocks T-cell activation and arrests cell proliferation. At least 2 esterified rapamycin derivatives that are orally available are in development.

side effects including rash, fatigue, mucositis, hematologic toxicity, and hyperglycemia.93 A recent trial comparing temsirolimus with IFN or temsirolimus/IFN has demonstrated a significant improvement in OS for patients with poor-risk RCC receiving temsirolimus alone.94

c-Met/Hepatic Growth Factor c-Met Pathway The c-Met protooncogene located at chromosome 7q31 encodes the met receptor, a receptor TK. Binding of the c-Met receptor ligand hepatocyte growth factor triggers autophosphorylation and activation of a downstream signaling cascade.95 The c-met protooncogene appears to have a role in diverse biologic activities, including cell motility, morphogenic differentiation, angiogenesis, mitogenesis, cell proliferation, and invasion. c-Met has been implicated in tumorigenesis of hereditary papillary renal carcinoma. It was also expressed in 80% of sporadic papillary RCCs and might predict aggressive tumor characteristics. Increasing tumor stage was associated with c-Met expression,96 and a trend toward longer OS was noted in patients with c-Met–negative tumors. c-Met is also overexpressed in clear-cell RCC cells and is phosphorylated in the absence of hepatic growth factor. In this report, c-Met was inhibited by transfection of wild-type VHL, without a decrease in expression level of c-Met protein.97 These findings suggest c-Met might be a relevant target in papillary and clear-cell RCC.

Therapeutic Approaches to c-Met Inhibition Several c-Met inhibitors are currently in clinical trials, including ARQ 197. ARQ 197 has shown in vivo and in vitro antitumor activity across a wide range of human tumor cell lines and in murine xenograft models of human colon, pancreas, and breast cancers. Currently, a phase I trial is in progress in patients with refractory solid tumors. The drug is administered orally at a starting dose of 10 mg twice daily with dose escalation up to 800 mg per day until recommended phase II dose is established. ARQ 197 will be given for 2 weeks followed by 1 week without therapy, repeating the cycle every 3 weeks.

Miscellaneous Targets c-Kit

Temsirolimus. Temsirolimus, or temsirolimus, binds to immunophilin FKBP-12 to form a complex that interacts with the mTOR, blocking its activity. This, in turn, results in inhibition of key signal transduction pathways including those regulated by p70s6 kinase and the eukaryotic initiation factor 4E-binding protein, resulting in cell cycle arrest at G1.89,90 RAD001. RAD001, or everolimus, is an orally bioavailable rapamycin analogue. It demonstrated dose-dependent antitumor activity in preclinical models and showed additive antiproliferative effects in cancer cell lines when used in combination with the EGFR TK inhibitor gefitinib.91,92 Both agents are now being investigated in phase II/III trials. In phase I/II trials, tumor regression was documented in patients with RCC. Temsirolimus treatment is well tolerated with

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c-Kit encodes the membrane-bound TK c-Kit. c-Kit has been reported to be a marker for chromophobic RCC and renal angiomyolipoma.98 The mRNA levels were increased by > 7-fold in clear-cell RCC tumor cells compared with normal kidney tissues. Immunohistochemical expression of c-Kit was not detected in most other types of renal cell tumors evaluated. The differential expression of c-Kit in these renal tumors might have diagnostic and therapeutic implications.

smac/DIABLO The intracellular apoptotic pathway has to be activated for cell death to occur. Signals inducing apoptosis can be very diverse and encompass the direct stimulation of death receptors or cellular stress induced by chemicals and irradiation. The ability to evade apoptosis might enhance the cells’ propensity to

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Mohamad K. Khasawneh, Ronald M. Bukowski malignancy. X-linked inhibitor of apoptosis protein (IAP) is a potent antiapoptotic factor that belongs to the IAP family. This factor has 3 identified inhibitors, one of which is the second mitochondria-derived activator of caspases/direct IAP-binding protein with low proteasome inhibitor (smac/DIABLO), which is localized in the cell mitochondria and released into the cytoplasm upon apoptotic stimuli.99 In an analysis of 66 RCC specimens, smac/DIABLO mRNA expression was found in all tumor stages, nuclear grades, and histologic subtypes. Increased x-linked IAP:smac/DIABLO ratio correlated with more progressive tumors (from pT1 to pT3), and lower expression ratio was seen in early stage (pT1 and pT2) compared with pT3 tumors.100 Patients with RCC expressing smac/DIABLO had a better prognosis and longer 5-year disease-free survival after radical nephrectomy.101 These findings suggest that smac/DIABLO plays an important role in the regulation of apoptotic responses in cancer cells to immuneand drug-mediated therapies.

T-Regulatory Cells Regulatory T cells (Tregs) induce immune tolerance by suppressing host immune responses against self or nonself antigens. Because cancer cells express self antigens, they escape recognition by Tregs. In RCC, Tregs with suppressive activity have been reported.102 Vaccination with RNA-transfected dendritic cells is an effective strategy to stimulate potent T-cell responses in patients with metastatic cancers.103,104 CD4+ T cells constitutively expressing the IL-2 receptor _-chain (CD25) act in a regulatory capacity by suppressing the activation and function of other T cells.105 Their physiologic role is to protect the host against development of autoimmunity by regulating immune responses against antigens expressed by normal tissue.106,107 Because tumor antigens are largely self antigens, Tregs might also prevent the tumorbearing host from mounting an effective antitumor immune response. Previous studies have shown that increased numbers of CD4+CD25+ Tregs can be found in patients with advancedstage cancer108 and that high Treg frequencies are associated with reduced survival.109 The important role of CD4+CD25+ Tregs in controlling tumor growth was further highlighted by the demonstration that depletion of Tregs using anti-CD25 antibodies can evoke effective antitumor immunity in mice.110,111 Moreover, antiCD25 therapy enhanced the therapeutic efficacy of granulocytemacrophage colony-stimulating factor and prolonged survival of tumor-bearing animals.112 Cumulatively, these experimental data suggest that the efficacy of cancer vaccination and other immunotherapeutic approaches could be enhanced by treatment with agents that preferentially deplete CD4+CD25+ Tregs. Compounds that target cells expressing the IL-2 receptor CD25 subunit might have this effect. In a study by Dannull et al, recombinant IL-2 diphtheria toxin conjugate denileukin diftitox (also known as DAB389IL-2) was used to eliminate CD25-expressing Tregs in patients with metastatic RCC.113 Denileukin diftitox contains the catalytic and membrane translocation domain of diphtheria toxin.114

The binding domain for the diphtheria toxin receptor, however, is deleted and replaced by the human IL-2 gene, which allows for targeting of CD25-expressing cells. The cytotoxic action of denileukin diftitox occurs as a result of binding to the high-affinity IL-2 receptor, subsequent internalization, and enzymatic inhibition of protein synthesis, ultimately leading to cell death. The study suggested that denileukin diftitox could selectively eliminate Tregs from human peripheral blood mononuclear cells in a dose-dependent manner without apparent bystander toxicity to other peripheral blood mononuclear cells or CD4+ T cells with intermediate- or low-level expression of CD25. Treg depletion resulted in enhanced stimulation of proliferative and cytotoxic T-cell responses in vitro but only when denileukin diftitox was used before and omitted during the T-cell priming phase. Depletion of Tregs in patients with RCC followed by vaccination with tumor RNA-transfected dendritic cells led to improved stimulation of tumor-specific T cells when compared with vaccination alone. This study provided evidence that in vivo elimination of Tregs is capable of enhancing the magnitude of vaccine-mediated, tumor-specific T-cell responses in humans. Denileukin is a difficult agent to use and has significant toxicity. Therefore, other approaches to deplete Tregs are needed. In a recent report,115 the Th1 and Th2 profiles and Treg (CD4+CD25+FoxP3+) levels in patients treated with sunitinib were determined at baseline and after 28 days of therapy. A significant reduction in Treg numbers on day 28 was noted and was accompanied by a change from a Th2 to a Th1 bias. All patients had tumor shrinkage including 2 patients with partial response, and this shrinkage correlated with the development of a Th1 bias. The underlying mechanism of this effect is under study, and it is possible that this immunostimulatory effect might contribute to the antitumor efficacy of sunitinib in metastatic RCC.

Conclusion The role of targeted therapy in patients with advanced-stage RCC is firmly established. Recent data indicate agents such as sunitinib, sorafenib, and temsirolimus produce significant clinical benefit. The identification of various targets in clear-cell and papillary RCC has facilitated this development and the investigation of these agents. Additionally, the role of these targets in predicting clinical behavior and outcomes with targeted therapy are under assessment. Patient selection for various treatments based on target expression at the protein or gene level should now be a major goal in translational studies accompanying development of novel treatment approaches in patients with advanced-stage RCC.

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Genitourinary Cancer Supplement

December 2006