Promising molecular targeted therapies in breast cancer.

In recent years, there has been a significant improvement in the understanding of molecular events and critical pathways involved in breast cancer. This has led to the identification of novel targets and development of anticancer therapies referred to as targeted therapy. Targeted therapy has high specificity for the molecules involved in key molecular events that are responsible for cancer phenotype such as cell growth, survival, migration, invasion, metastasis, apoptosis, cell-cycle progression, and angiogenesis. Targeted agents that have been approved for breast cancer include trastuzumab and lapatinib, directed against human epidermal growth factor receptor 2 (HER2) and bevacizumab, directed against vascular endothelial growth factor (VEGF). Several other targeted agents currently under evaluation in preclinical and clinical trials include inhibitors of epidermal growth factor receptor (EGFR), dual EGFR and HER2 inhibitors, VEGF/VEGFR inhibitors, and agents that interfere with crucial signaling pathways such as PI3K/AKT/mTOR and RAS/MEK/ERK; agents against other tyrosine kinases such as Src, insulin-like growth factor (IGF)/IGF-receptor (IGFR); agents that promote apoptosis such as Poly ADP ribose polymerase inhibitors; agents that target invasion and metastasis such as matrix metalloproteinases inhibitors and others. In this review, we highlight the most promising targeted agents and their combination with mainstream chemotherapeutic drugs in clinical trials.

Introduction

Breast cancer is the most frequent cancer among women with an estimated 1.38 million new cancer cases diagnosed in 2008 (23% of all cancers), and ranks second overall (10.9% of all cancers) worldwide. In United States, incidence and mortality due to breast cancer was 152 and 40 cases, respectively, per 100,000 in the year 2008. A decline in incidence of new breast cancer cases has been observed in USA and several developed countries. Several factors attributed for this trend, include changes in reproductive factors, rates of mammography screening, environmental exposures, changes in diet, and reduced use of hormone replacement therapy (HRT). On other hand, the incidence of breast cancer in India is on the rise with 115 cases and 53 deaths per 100,000 annually. This rise is probably due to lifestyle changes in women and lack of awareness programs, and it is rapidly becoming the number one cancer in females.[1] The two most common types of breast cancer are ductal and lobular carcinoma named after their origin in breast tissue. Ductal carcinomas make 85% to 90%, and whereas, 8% are lobular breast cancers. Others types include invasive (infiltrating) and inflammatory breast cancer. Tumors with estrogen receptor (ER)-positive and/or progesterone receptor (PR)-positive are known to have lower risks of mortality after diagnosis compared to women with ER- and/or PR-negative disease. Several clinical studies have demonstrated women with hormone receptor (HR)-positive tumors have survival advantage by treatment with adjuvant hormonal and/or chemotherapeutic regimens.[2]

Breast cancer is a heterogeneous disease with a variety of pathological entities and varied clinical behavior and different molecular alterations driving its growth, survival, and response to treatment. Chemotherapeutic drugs available for treatment of breast cancer can be divided into alkylating agents (cisplatin, carboplatin, and cyclophosphamide), anthracyclines (doxorubicin, epirubicin, and idarubicin), plant alkaloids (paclitaxel, vinorelbine, and vindesine), and topoisomerase inhibitors (irinotecan, etoposide, and teniposide). Despite significant advances in early detection and steady progress in the treatment with systemic agents, most breast cancers develop resistance to drugs. In recent years, understanding of the underlying biological mechanisms of carcinogenesis and the altered molecular events has led to the identification of novel molecular targets and development of targeted therapies. Targeting the pathways that promote or sustain growth and invasion of carcinoma cells is critical to effective treatment of breast cancer. In the past two decades, several monoclonal antibodies and small-molecule inhibitors [Figure 1] have been developed and tested in clinical trials that target cancer characteristics such as cell growth, survival, angiogenesis, and metastasis. Several of these targeted agents significantly improved the survival and outcome of the breast cancer patients. One of the earliest and most crucial cornerstone development in the field of targeted therapy was trastuzumab, a monoclonal antibody (Mab) against epidermal growth factor receptor 2 (HER2) overexpressing tumors and proved effective in treating women with HER2-positive breast cancer. In more recent times, lapatinib a selective reversible inhibitor of both HER1 and HER2 and bevacizumab, a Mab against vascular endothelial growth factor (VEGF) have also been successful in improving cure rates, quality of life, and disease prevention in cancer patients. Moreover, targeting both HER2 with trastuzumab and VEGF with bevacizumab in combination with chemotherapy has become a further milestone of molecular targeted therapy in breast cancer.[3] With more clinical trials, it is becoming obvious that multiple targets regulating growth of cancer cells may need to be inhibited for optimal efficacy and reduced resistance.

Figure 1

Overview intracellular signal transduction pathways involved in the proliferation and progression of breast cancer. Targeted therapy agents discussed in this review and their main inhibition targets are illustrated. EGF: Epidermal growth factor; EGFR: EGF receptor; HGF: hepatocyte growth factor; c-MET: mesenchymal– epithelial transition factor; PDGF: Platelet-derived growth factor; PDGFR: PDGF receptor; IGF-1: insulin-like growth factor-I; IGF-1R: IGF-1 receptor; PI3K: phosphatidylinositol 3-kinase; Ras: Rat sarcoma subfamily of GTPases; AKT: protein kinases B; PDK1: pyruvate dehydrogenase kinase isozyme 1; mTOR: mammalian target of rapamycin; MEK: mitogen-activated protein kinase kinase; VEGF: vascular endothelial growth factor; VEGFR: VEGF receptor; BRAF: B-type RAF kinase; src: v-Src (Rous sarcoma virus) tyrosine kinase; BCRABL: Philadelphia chromosome; JAK/STAT: Janus kinases/signal transducers and activators of transcription; PTEN: phosphatase and tensin homolog; HDAC: histone deacetylases.

Other target agents that have evolved in recent times include tyrosine kinases such as Src, insulin-like growth factor (IGF)/IGF-receptor (IGFR); agents that interfere with critical pathways, such as PI3K/AKT/mTOR inhibitors, RAS/MEK/ERK inhibitors; agents that promote apoptosis such as Poly ADP ribose polymerase (PARP) inhibitors; agents that target invasion and metastasis such as MMP inhibitors, and others. Several of trastuzumab promising targeted agents are still in preclinical and early clinical trials. Herein, we review the most current development and information that have shaped the targeted therapy for breast cancer.

Targeting Epidermal Growth Factor Receptor Family

Epidermal growth factor receptor (EGFR) is a family of transmembrane growth factor receptor tyrosine kinases involved in regulation of cell proliferation and survival of epithelial cells. EGFR family includes four receptors: EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4. EGFR and HER2 are overexpressed in approximately 40% and 25% of breast cancers, respectively, and are associated with aggressive clinical behavior and poor prognosis. Preclinical studies suggested that inhibiting this target might have anti-tumor activity and reverse chemoresistance.

EGFR Inhibitors

Cetuximab (Erbitux) is a Mab that competitively binds with the extracellular domain of EGFR and prevents the receptor from activating EGF ligand, thereby inhibiting intracellular signal transduction. Preclinical studies demonstrated synergistic effect of cetuximab with paclitaxel in breast cancer models, but it failed to show efficacy in a phase I study with MBC patients.[4] In the recent mid-stage study results presented at the European Society for Medical Oncology Congress, cetuximab in combination with cisplatin was indicated to significantly reduce the risk of disease progression in heavily pretreated women with metastatic triple negative breast cancer. Cetuximab plus cisplatin increased median time of progression-free survival (PFS) to 3.7 months compared to 1.5 months by cisplatin alone.[5] A phase II trial is currently testing efficacy of cetuximab alone and in combination with carboplatin in metastatic triple negative breast cancer (TNBC). Other ongoing clinical trials with cetuximab include combination with agents such as irinotecan, bortezomib, ixabepilone, and trastuzumab in triple negative locally advanced non-resectable and/or MBC.

Gefitinib is a small molecule that reversibly inhibits EGFR tyrosine kinase autophosphorylation and inhibits downstream signaling. Several clinical studies of single agent gefitinib and gefitinib plus chemotherapy or hormonal therapy for breast cancer have been done. Single agent studies with EGFR inhibitor gefitinib resulted in low clinical benefit rate (CBR) and no tumor response in advanced breast cancer (ABC) patients either with hormone resistance or with hormone receptor (HR)-negative tumors.[6] In a recent randomized study, anastrozole plus gefitinib was associated with marked improvement in PFS (14.3 months) compared with anastrozole plus placebo (8.4 months).[7] Several recently completed clinical trials have studied gefitinib in combination with chemotherapeutic agents, including trastuzumab, docetaxol, and tamoxifen. In an ongoing study, gefitinib is being tested in combination of arimidex, faslodex, and iressa to determine the clinical response rate whereas, a second study focuses on the clinical benefit (CB) of anastrozole and gefitinib vs. fulvestrant and gefitinib in postmenopausal women with recurrent or metastatic hormone receptor-positive breast cancer. Gefitinib was found to block the acquired tamoxifen resistance mediated through EGFR signaling.

Erlotinib is another orally active EGFR tyrosine kinase inhibitor (TKI). In a phase I study combination of erlotinib and trastuzumab was well tolerated and showed preliminary evidence of anticancer activity.[8] An overall response rate of 67% was observed in MBC patients treated with erlotinib in combination with capecitabine and docetaxol.[9] Many other studies of erlotinib combined with bevacizumab, docetaxel, vinorelbine, and capecitabine have been reported with mixed responses.

HER2 Inhibitors

Trastuzumab (commonly referred to as herceptin) was the first recombinant bivalent humanized Mab targeted against extracellular domain of HER2 reported in 1998, which blocks intracellular signaling. Trastuzumab was shown to inhibit tumor cells overexpressing HER2 both in vitro and in vivo. Unlike chemotherapy, trastuzumab did not have toxic effects such as nausea, vomiting, hair loss, and bone marrow toxicity. Although cardiotoxicity events are concerns with trastuzumab these effects appear to be mostly treatable and reversible. Anti-HER2 therapies have significantly improved the clinical outcome of aggressive tumors. Large multicentre randomized trials revealed effectiveness of trastuzumab as adjuvant therapy when added in sequential or concurrently with taxane-based chemotherapy in HER2-positive early and advanced MBC.

HERCULES trial evaluated herceptin, cyclophosphamide, and epirubicin in MBC and reported high tumor response with low cardiotoxicity.[10] Clinical trials have also confirmed that combining trastuzumab with anthracycline-taxane-based neo-adjuvant chemotherapy in patients with HER2-positive breast cancer will result in high pathologic complete response (pCR) rate.[11] NOAH trial reported the benefit of one year trastuzumab therapy in significant improvement of 3-year event-free survival (EFS) in patients with HER2-positive breast cancer by 71% compared to 56% without trastuzumab.[12]

In recent studies, the combination of trastuzumab, docetaxel, and capecitabine or vinorelbine was highly active as first-line combination therapy for HER2-positive locally advanced or MBC. The combination of capecitabine and trastuzumab was active and well-tolerated in patients with HER2-overexpressing breast cancer resistant to both anthracyclines and taxanes with median overall survival (OS) and PFS as 22.3 and 4.1 months, respectively.[13] On the other hand, trastuzumab plus capecitabine and docetaxel regimen as first-line therapy was also found highly effective with an median OS of 25.5 months, and PFS of 7.8 months in anthracycline-pretreated patients with HER-2-overexpressing breast cancer.[14]

The synergism of trastuzumab and vinorelbine led a phase II trial with weekly vinorelbine/trastuzumab and proved to be an active and safe regimen in patients with HER2-positive MBC with an unfavorable prognosis.[15] Trastuzumab was also found to be promising with low toxicity when given concurrently with whole brain radiotherapy in patients HER2-positive patients with brain metastases. Trastuzumab combined with cytotoxic agents presents encouraging results in MBC, but cardiac toxicity has limited some combinations. Therefore, it is necessary to evaluate the optimal combination of trastuzumab with other agents and also to develop alternative targeted agents.

EGFR and HER2 Dual Inhibitors

Despite initial efficacy, a large number of patients with MBC develop acquired resistance to trastuzumab. Moreover, the inability of trastuzumab to cross blood-brain barrier has been a major reason behind brain metastasis in patients on trastuzumab therapy. A viable option to improve trastuzumab performance is to combine them with other signal transduction pathways inhibitors. Therefore, small molecule tyrosine kinase inhibitors (TKI) that target HER2 overexpressing tumors and cross blood-brain barrier could show activity against brain metastases. Dual EGFR-HER2 inhibitors for breast cancer include lapatinib, cetuximab, pertuzumab, canertinib, and neratinib.

Lapatinib is an oral dual TKI that targets both EGFR and HER2, by blocking the downstream signaling pathways from these receptors. Lapatinib has been extensively studied in multiple clinical settings for treatment of patients with advanced or MBC. Studies have demonstrated promising clinical activity of lapatinib monotherapy in previously untreated patients and those progressed on trastuzumab-containing therapy. Despite disease progression on prior trastuzumab-based therapy, lapatinib in combination with trastuzumab significantly improved PFS and CBR versus lapatinib alone, thus offering a chemotherapy-free option with an acceptable safety profile to patients with ErbB2-positive MBC. The most common treatment-related adverse events were rash, diarrhea, nausea, and fatigue.[16] A subgroup of HER2-overexpressing breast tumors were found to co-express p95HER2, which is responsible for intrinsic trastuzumab resistance. Lapatinib monotherapy or in combination with capecitabine was found effective in patients with p95HER2-positive and p95HER2-negative HER2-positive breast tumors.[17]

The Lapatinib Expanded Access Program (LEAP) was designed to provide access to lapatinib plus capecitabine for HER2-positive MBC patients previously treated with anthracycline, taxane, and trastuzumab and had no other treatment options. The median PFS and OS were 21.1 and 39.6 weeks, respectively. Further subgroup analysis showed longer PFS and OS in patients who had not received prior capecitabine. These results demonstrated the safety and efficacy of lapatinib in a broader patient population compared with a clinical trial.[18]

One third of patients with advanced HER2-positive breast cancer develop brain metastasis. In clinical studies, lapatinib regimens showed modest response to therapy with promising activity against brain metastasis. A phase II study confirmed modest regression of central nervous system (CNS) lesion by lapatinib and was associated with volumetric reduction, improvement in PFS, and neurologic signs and symptoms. Additional responses were observed with the combination of lapatinib and capecitabine volumetric reduction in their CNS lesions.[19] In another recent study, lapatinib combined with capecitabine was concluded as an active treatment option for women with refractory HER2-positive MBC, including those with progressive CNS disease.[20]

Lapatinib plus hormonal agents such as letrozole are being studied in patients with resistance to hormonal therapy. Eighty percent of EGFR-positive patients exhibited response to lapatinib. These promising results of lapatinib on MBC and low cardiotoxicity, encourage use of lapatinib as adjuvant therapy in early HER2-positive breast cancer and HER2-negative tumors that express one or more ErbB family members.

Afatinib (BIBW-2992) an anilino-quinazoline derivative is an oral dual receptor TKI of EGFR and HER-2/neu. Afatinib was found to be active against tumors overexpressing EGFR with the secondary Thr790Met point mutation, which confers resistance to the first-generation EGFR inhibitors gefitinib and erlotinib.[21] Currently, several phase I/II trials are underway to explore the efficacy of afatinib alone versus with lapatinib and trastuzumab in patients with HER2-positive treatment-naïve Stage IIIa locally ABC. An open-label, randomized phase III study ′LUX-Breast I′ is currently enrolling HER2-positive patients with ABC previously treated with trastuzumab. The study investigates whether treatment with afatinib can extend PFS as compared to trastuzumab when both are added to the standard chemotherapy treatment with vinorelbine. Overall survival, tolerability, and safety will also be assessed in the study.

Perutuzumab is a Mab that binds to a different epitope of HER2 than trastuzumab and sterically blocks both HER2 homo- and heterodimerisation with other ErbB receptors. Single agent trials with perutuzumab were well tolerated without major adverse events. In phase II trial, unselected patients with HER2-negative disease pertuzumab monotherapy had limited efficacy.[22] On the other hand, perutuzumab and trastuzumab are known to have complementary mechanisms of action and results in enhanced antitumor activity. In recent phase II clinical trial, efficacy and safety profile of combination therapy of trastuzumab plus perutuzumab was assessed in patients with HER2-positive breast cancer. This combination was well tolerated and improved the median PSF of 5.5 months in patients who experienced progression during prior trastuzumab therapy.[23]

Nertinib is an orally administered irreversible pan-HER TKI. Preliminary studies have demonstrated antitumor activity of nertinib in patients previously treated with trastuzumab, anthracyclines, and taxanes.[24] In a recent clinical trial, oral neratinib demonstrated substantial clinical activity and was well tolerated among both heavily pretreated and trastuzumab-naïve ErbB2-positive ABC patients. The 16-week PFS rates were 59% in patients pretreated with trastuzumab and 78% in patients with no prior trastuzumab treatment. The median PFS were 22.3 and 39.6 weeks, with objective response rates of 24% and 56% among patients with prior trastuzumab treatment and trastuzumab-naïve cohort, respectively. The most common adverse events were diarrhea, nausea, vomiting, and fatigue.[25] Several randomized phase III trials are now going on with nertinib in MBC and in adjuvant settings.

Canertinib (CI-1033) is also an irreversible pan-HER TKI that covalently binds to the ATP-binding site of the intracellular kinase domain. In addition, it blocks the highly tumorigenic, constitutively activated variant of erbB-1, EGFRvIII, and inhibits downstream signaling through both the Ras/ MAP kinase, and PI-3 kinase/AKT pathways. Canertinib being an irreversible inhibitor; provides prolonged suppression of erbB receptor-mediated signaling. Preclinical data show CI-1033 to be efficacious against a variety of human tumors in mouse xenograft models, including breast carcinomas.[26] So far clinical results have not been very encouraging. However, several phase II trials in MBC are underway to determine the antitumor activity of this drug. Other anti-HER tyrosine kinase inhibitors that are currently being studied include HKI-272 and EKB-569 and show early evidence of clinical benefits.

Targeting Vascular Endothelial Growth factor family

In breast cancer, angiogenesis plays an important role in growth, invasion, and metastasis. Vascular Endothelial Growth factor (VEGF) is a potent inducer of cell migration, invasion, vascular permeability, and vessel formation. Five related glycoproteins namely, VEGFA, VEGFB, VEFGC, VEGFD, and placental growth factor (PGF) act via three receptor tyrosine kinases VEGFR-1, VEGFR-2, and VEGFR-3. Overexpression of VEGF has been associated with poor clinical outcomes in patients with both lymph node-positive and -negative breast cancers.[27] Agents that block VEGF have shown to inhibit tumor angiogenesis and growth of breast cancer. VEGF is inhibited in several ways with agents that bind to the ligand (bevacizumab), inhibit VEGF receptor tyrosine kinase (sunitinib), inhibit downstream effectors of VEGF activity (mammalian target of rapamycin (mTOR) inhibitors), and agents that modulate VEGF production (HER2-targeting agents), etc.

VEGF Inhibitors

Bevacizumab, a humanized Mab directed against all isoforms of VEGFA. In E2100 randomized trial, bevacizumab plus paclitaxel achieved OR rate of 48.9% vs. 22.2% in paclitaxel alone group when given as first-line therapy in patients with MBC. This study demonstrated effectiveness of bevacizumab in inhibiting angiogenesis in breast cancer.[28] In another similar randomized trial AVADO patients received docetaxel alone or in combination with bevacizumab as the first-line treatment of HER2-negative MBC.[29] Both the trials showed significant increase in PFS rate among patient receiving bevacizumab. Bevacizumab is also being investigated as maintenance therapy in triple receptor-negative breast cancer. It is added as the standard adjuvant therapy with doxorubicin/cyclophosphamide (AC) and paclitaxel in HER2-negative breast cancer patients. In a study, the triple combination of bevacizumab, capecitabine, and docetaxel as neoadjuvant therapy was evaluated for breast cancer. The results demonstrated a 22% pCR rate in a HER2-negative population thus suggesting that addition of bevacizumab increases the activity of neoadjuvant capecitabine-docetaxel.[30]

In HER2-positive breast cancer patients combining bevacizumab with trastuzumab showed encouraging results. In recent phase III trials, standard chemotherapy was compared with chemotherapy plus bevacizumab, and chemotherapy with docetaxel, carboplatin and trastuzumab with or without bevacizumab in HER2 overexpressing breast cancer. These studies were promising and provided insights regarding use of bevacizumab as targeted therapy in HER2-positive breast cancer.[31] A meta-analysis of randomized controlled trials showed that the addition of bevacizumab to chemotherapy could offer meaningful improvement in PFS and OR rates in patients with MBC.[32] Bevacizumab is being tested as first-line treatment in combination with docetaxel, capecitabine, anthracyclines, gencitabine, letrozole, and paclitaxel in ABCs.

Aflibercept (VEGF Trap) is a fusion protein that binds to all forms of VEGF-A. In addition, aflibercept also binds PGF which is also implicated in breast tumor angiogenesis. Although aflibercept was found to inhibit tumor growth and metastasis in murine tumor models,[33] clinical trials failed to show promising results. Currently, phase II trial is underway with VEGF trap in patients with MBC previously treated with anthracycline and/or taxane.

Multi-kinase Inhibitors

Another approach to block angiogenesis is to target and inhibit multiple members of receptor tyrosine kinases known to play role in tumor proliferation and neovascularization. There are several small molecules that act as multi-kinase inhibitors.

Sunitinib (SU11248) is an oral multi-TKI that targets VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-α and β, stem-cell factor receptor (c-KIT), and fms-like tyrosine kinase receptor 3 (flt-3). These targets play important role in the growth and survival of breast cancer. Preclinical studies demonstrated inhibition and regression of breast cancer tumors in animal models. In phase II study, sunitinib monotherapy was well tolerated and was found active in patients with heavily pretreated MBC. The common adverse events were fatigue, nausea, diarrhea, mucosal inflammation, and anorexia.[34]

Sunitinib has also been evaluated in clinical trials in combination with chemotherapeutic agents. A phase II study evaluated paclitaxel and bevacizumab with or without sunitinib as first-line treatment in heavily treated MBC, but was prematurely terminated due to toxicity effects such as neutropenia, thrombocytopenia, fatigue, diarrhea, and nausea.[35] Currently, other phase II clinical trials are investigating sunitinib as single agent in pretreated patients with taxane and in combination with docetaxel or trastuzumab. Although studies so far with sunitinib have given mixed results, however, it would be worth exploring strategies that inhibit both VEGFR and VEGF.

Semaxinib is a TKI targeting VEGFR2, PDGFR-β and fibroblast growth factor receptor 1 (FGFR1). Preclinical xenograft studies in animals with these agents showed promising antitumor activity; however, they failed to show desirable response in clinical trials. In a study, semaxinib and doxorubicin showed evidence of an unfavorable cardiac interaction and thus prohibited further investigation of this combination.[36]

Pazopanib, a multitargeted tyrosine kinase inhibitor selectively inhibits VEGF-mediated endothelial cell proliferation by targeting VEGFR1, VEGFR2, and VEGFR3. Pazopanib exhibited in vivo and in vitro activity against tumor growth. In MBC, the combination of pazopanib with lapatinib was more effective than lapatinib alone.[37] Ongoing phase II trials are comparing pazopanib plus lapatinib to lapatinib alone in patients with advanced or MBC and inflammatory breast cancer. Currently, pazopanib is also being evaluated as a single agent and in combinational therapy in several phase II and III trials.

Axitinib is a novel TKI that targets all VEGFR isoforms, PDGFR and c-KIT. Axitinib was well tolerated in a phase I study with MBC patients. The frequent side effects include fatigue, hypertension, diarrhea, hand-foot syndrome, and proteinuria. Axitinib is considered as first-line combination due to its antiangiogenic and antitumor activity with acceptable safety profile. Phase II study of axitinib with docetaxel in MBC patients with no prior chemotherapy had longer PFS compared to docetaxel plus placebo.[38] Several larger, randomized phase II/III studies are underway to prove the efficacy of this TKI as a single- agent and in combination with other chemotherapeutic agents.

Vandetanib (ZD6474) is a TKI that targets VEGFR2 and EGFR to inhibit angiogenesis and tumor growth. Vandetanib monotherapy was generally well tolerated but had limited activity in patients with refractory MBC.[39] Clinical studies are underway to confirm its activity as a single-agent and in combination with chemotherapeutic agents. Ongoing phase II and III clinical trials will better define the appropriate schedule, the optimal setting of evaluation, and the safety of long-term use of vandetanib.

Targeting RAS/MEK/ERK Pathway

The Ras superfamily of GTPases are crucial regulatory switches involved in cell proliferation and differentiation. Oncogenic Ras mutation occurs less than 5% but overexpression of Ras protein has been described in breast cancer. Overexpression of Rac and Rho proteins has been associated with increased invasiveness and MBC. Ras proteins are stimulated by several growth factor receptors involved in breast cancer such as HER1, HER2, IGF-1, and ERα. Ras in turn activates Raf/MEK/ERk and PI3K/Akt kinase cascades that are involved in cell survival and proliferation.

Farensyltransferase Inhibitors

The enzyme farensyltransferase is involved in post-translational modification of Ras thereby making it active for signal transduction. It is therefore logical to inhibit Ras modification and downstream signaling as a viable therapeutic target with farensyltransferase inhibitors (FTIs). FTIs are thought to have antitumor activity beyond Ras pathways as they also act on several other proteins involved in cell regulation that require farnesylation for their activity.

Tipifarnib is an oral FTI that inhibits farnesylation of Ras and other proteins involved in signal transduction pathways. Tipifarnib has shown in vitro and in vivo antitumor activity in preclinical studies. Single-agent phase I study was well tolerated without significant toxicity effects. In a phase II trial, tipifarnib demonstrated 10% partial response and clear CB in ABC patients.[40] Major side effects include neutropenia, thrombocytopenia and neurotoxicity. Tipifarnib combined with doxorubicin and cyclophosphamide in ABC patients resulted inhibition of FTase activity by 91% in human breast tumors in vivo and down-regulated p-STAT3 by 77% and improved the breast pCR rate (25%).[41] Several trials in breast cancer have been completed with FTIs alone or in combination with endocrine or cytotoxic therapy.

R115777 and SCH66336 are other FTIs that have entered clinical trials and have shown safety and favorable therapeutic indices. Studies evaluating combination of trastuzumab and R115777 are being pursued. Taxane and FTI combinations are also being evaluated because preclinical studies suggest that these classes of anticancer agents may be synergistic.

Raf Inhibitors

Raf is a downstream effector of Ras, which is phosphorylated and activates mitogen activated protein kinase (MAPK) cascade. Raf exists in three isoforms namely, A-Raf, B-Raf, and Raf-1. Mutated B-Raf has been reported in at least 7% of human cancers.

Sorafenib (Bay 43-9006) is a small molecule inhibitor of Raf kinase activity along with other targets such as VEGFR1, VEGFR2, VEGFR3, PDGFR-β, c-kit, and flt3, thereby simultaneously blocks cell proliferation, angiogenesis and causes apoptosis. In a phase II study with MBC, sorafenib achieved stable disease, suggesting that sorafenib modified tumor growth rate instead of reducing tumor volume. Therefore, sorafenib combinations with standard therapy have been suggested with end points more sensitive to effects of targeted agents, such as disease stabilization.[42] More frequent adverse effects include skin rash, hand-foot syndrome, hypertension, and diarrhea. In patients with MBC that had progressed after at least one prior chemotherapy regimen, oral sorafenib showed minimal improvement to treatment. Further investigation of single-agent sorafenib in this patient population is discouraged. However, sorafenib in combination with capecitabine significantly improved PFS in patients with breast cancer from 4.1 to 6.4 months.[43] Currently, a phase III trial is comparing capecitabine in combination with sorafenib or placebo in the treatment of locally advanced or metastatic HER2-negative breast cancer.

MEK Inhibitors

CI-1040 is a small molecule inhibitor of mitogen-activated Protein/Extracellular Signal-regulated Kinase (MEK) necessary for cancer cell growth. In phase I study with advanced malignancies, 28% patients achieved stable disease of 5.5 months with 46%-100% inhibition of tumor pERK. PD 0325901, a second generation MEK inhibitor, was found to significantly improve pharmacologic and pharmaceutical activity and reduced pERK in tumor tissue of patients with advanced cancer.[44]

Targeting Cell Cycle and Apoptosis

PI3K/Akt/mTOR Pathway Inhibitors

The PI3K/Akt pathway plays central role in a variety of cellular functions including proliferation, growth, survival, and angiogenesis. Uncontrolled activation of this pathway by ER, IGF-1, and HER3 is behind the development and progression of breast cancer. Oncogenic Ras and loss of PTEN are other stimulators of this pathway. Akt signaling is also known to regulate the serine-threonine kinase, and mammalian target of rapamycin (mTOR). Approximately 40% of primary breast cancers are known to have activating mutation of PI3K. Rapamycin targets mTOR and results in G1 arrest. Rapamycin derivatives such as everolimus, temsirolimus, and deforolimus have been studied in breast cancer.

Letrozole inhibits key molecules in the PI3K pathway that are important targets of new drugs being developed to overcome resistance. In a randomized phase II study, letrozole in combination with temsirolimus or alone in patients with HER2-positive MBC resulted in longer PFS than letrozole alone.[45] ER-positive breast tumors treated with everolimus showed a significant reduction in genes involved with proliferation and serve as markers of response and predicted patients who will derive most benefit from mTOR inhibition. In a recent in vitro study, another PI3K/AKT/mTOR blocker NVP-BEZ235 was found to show effect with pelitinib and canertinib in producing growth inhibition in cancer cells resistant against first and second generation (irreversible) ErbB-targeting drugs.[46] Randomized phase II/III trials of aromatase inhibitors combined with mTOR blockers are in progress to determine whether combined therapy can significantly prolong time to disease progression compared to endocrine therapy alone.

Cyclin-dependent Kinase Inhibitors

Flavopiridol is the Cyclin-dependent Kinase (CDK) inhibitor and is in clinical trials as an antitumor drug. Flavopiridol was found to sensitize breast cancer cells in vitro to TRAIL-induced apoptosis by facilitating early events in the apoptotic pathway. In phase I trial of docetaxel, followed by flavopiridol demonstrated encouraging clinical activity even in patients heavily pretreated with taxane and in patients with gemcitabine-refractory metastatic pancreatic cancer.[47] A phase I clinical trial testing combination of trastuzumab and flavopiridol in treating patients with MBC has recently been completed with results yet to be published.

Bcl-2 Silencing

Bcl-2 protein confers resistance to chemotherapy-mediated apoptotic signals in cancer. Oblimersen (G3139) is an antisense oligodeoxynucleotide that targets Bcl-2 mRNA and down-regulates Bcl-2 protein translation thereby enhancing antitumor effects of chemotherapy. Preclinical data showed enhancement of breast cancer cell death when G3139 was combined with anthracyclines and taxanes. In an efficacy and safety study, G3139, in combination with doxorubicin and docetaxel, was well tolerated. However, no pathologic complete response and little down-regulation of Bcl-2 in primary tumors were observed.[48]

p53

p53 tumor suppressor gene mutation is responsible for several human cancers, including breast cancers. Mutant p53 (mtp53) protein confers resistance to chemotherapeutic drugs and promote tumor cell survival. Therefore, restoring p53 function is a valid strategy to promote apoptosis of tumor cells. PRIMA-1 (p53 re-activation and induction of massive apoptosis) is a non-toxic small molecule that converts mtp53 to the active conformation and induces apoptosis in tumor cells. In an in vitro study, treatment of mtp53-expressing breast cancer cells with PRIMA-1 resulted in dose-dependent death via mitochondrial-dependent apoptotic pathway. However, cells expressing wtp53 protein were not affected. Thus, PRIMA-1 increased the susceptibility of mtp53-expressing breast tumors to apoptosis.[49] Preclinical animal study also indicated that PRIMA-1-caused tumor regression was well correlated with conversion of mutant to wild-type p53 conformation. Thus, PRIMA-1 has a potential to be used as a single agent or in combination with other anti-cancer compounds for treatment for human breast cancer.[50]

Targeting Invasion and Metastasis

Matrix Metalloproteinases Inhibitors

Matrix metalloproteinases (MMPs) are involved in tumor invasion and metastasis and have been implicated in breast, ovarian, colorectal, and lung cancer growth. In a phase I study, BAY 12-9566 (tanomastat), an inhibitor of MMP-2, MMP-9, and MMP-3, was well tolerated in patients with solid tumors and had no musculoskeletal effects.[51] Other studies have tested BAY 12-9566 in combination with doxorubicin, etoposide, and carboplatin and 5-fluorouracil and leucovorin. Currently, clinical trials are underway with MMP inhibitors such as marimastat, prinomastat, solimastat, BMS 275291, metastat, and neovastat.

Inhibition of the uPA System

The serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are often elevated in malignant tumors; hence, inhibition of this tumor-associated plasminogen activation system provides an attractive target for therapeutic strategies. WX-UK1, a novel 3-amidinophenylalanine-based inhibitor of the uPA system, was found to inhibit the invasive capacity of carcinoma cells in vitro. This led to a preclinical animal study, which also confirmed the activity of WX-UK1 as a single agent against breast tumor growth and metastasis.[52] Recently, a phase I clinical trial was successfully completed with WX-UK1 in combination with capecitabine in advanced malignancies.

Targeting src-family Tyrosine Kinases

src Inhibitors

The v-Src (Rous sarcoma virus) tyrosine kinase is a non-receptor associated with development, progression, and metastasis. Src is downstream of several growth factor receptors such as EGFR, PDGFR, IGF-1R, and HGFR. Src activation is observed in up to 40% of ER-positive cancers and is known to be responsible for antiestrogen resistance.

Dasatinib is a small-molecule inhibitor of Src-family kinases and protein kinases including Bcr-Abl, c-Kit, EphA2, and PDGF-β. It was found to be effective against basal-like breast cancers cells. In vitro and early clinical trials showed modest effect as a single-agent in triple-negative MBC patients.[53] There are currently several studies evaluating dasatinib as monotherapy or in combination with chemotherapy in breast cancer. CA180-004-phase I study evaluated escalating doses of capecitabine and dasatinib and demonstrated PR in 13.5% patients and few others had stable disease of ≥6 months.[54] Dasatinib in combination with paclitaxel also resulted in responses and/or prolonged stable disease in patients with prior taxane treatment.

Saracatinib (AZD-0530) is a highly selective, dual-specific small-molecule inhibitor of Src kinase and Bcr-Abl. In a recent preclinical study, saracatinib combined with fulvestrant was more effective in inhibiting proliferation of ER-positive breast cancer cells in vitro and in vivo than either drug alone. This study also demonstrated that saracatinib monotherapy led to drug resistance by bypass activation of the mTOR pathway. Therefore, use of mTOR kinase inhibitors with saracatinib may be a viable option to overcome drug resistance and increase efficacy than saracatinib alone.[55] Currently, studies are underway with saracatinib in combination with anastrozole in HER2-positive breast cancer patients.

Another oral src inhibitor bosutinib (SKI-606) was also found to inhibit phosphorylation of Src in mammary tumors thus preventing not only the appearance of palpable tumors but also stopped tumor growth in animals with pre-existing tumors. SKI-606 was found to control the development of mammary tumors by inducing differentiation. In a study, bosutinib monotherapy resulted in 6% PR, and 21% showed stable disease of ≥24 weeks. The median PFS was 15 weeks in these patients with pretreated MBC.[56] Current trials are evaluating the Src inhibitors in combination with endocrine agents in patients with evidence of endocrine resistance and also with chemotherapeutic agents.

HSP90 Inhibitors

Heat shock proteins (HSP) are vital to cell survival under conditions of stress. HSP inhibitors are novel agents that exert pro-apoptotic effects through inhibition of ATP binding on the ATP/ADP-binding pocket of the HSP. Initial HSP90 inhibitors, such as geldanamycin and tanespimycin (17-AAG), had limitations of solubility and hepatotoxicity. Tanespimycin in phase I study was well tolerated in combination with trastuzumab and was found to inhibit HSP90 function in patients with HER-2-positive breast cancer whose tumors have progressed during treatment with trastuzumab.[57] Retaspimycin (IPI-504), a derivative of geldanamycin and 17-AAG, is water soluble and was generally well tolerated in phase I/II trials, in non-small cell lung cancer and gastrointestinal stromal tumor. Phase I/II trials are currently underway to evaluate the dose and activity of IPI-504 in breast cancer.

Insulin-like Growth Factor Inhibitors

The IGF and IGF-IR plays a major role in cancer cell proliferation, survival and confers resistance to cytotoxic, hormonal, and targeted therapies in diverse tumor types, including breast cancer. Therapeutic strategies targeting the IGF-IR may therefore be effective broad-spectrum anticancer agents. IMC-A12, a fully human monoclonal IgG1 antibody binds with high affinity to the IGF-IR and prevents ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates internalization and degradation of the IGF-IR. In preclinical animal studies IMC-A12 was found to inhibit growth of a variety of tumors, including breast, lung, colon, and pancreas.[58] Currently, a clinical trial is testing IMC-A12 with antiestrogen such as tamoxifen, anastrozole, letrozole, exemestane or fulvestrant to treat breast cancer. Other ongoing studies have used IMC-A12 in combination with cixutumumab and temsirolimus or capecitabine and lapatinib to treat MBC. Results so far have shown promising activity when combined with cytotoxic agents or other targeted therapeutics.

Nordihydroguaiaretic acid (NDGA) is a phenolic compound that inhibits two receptor tyrosine kinases, namely, IGF-1R and HER2. In vitro studies suggested that NDGA induced DNA fragmentation, cleavage of -PARP and caspase-3. Combination treatment of NDGA and trastuzumab resulted in greater efficacy in trastuzumab-refractory cells than either agent alone, suggesting that NDGA increases the sensitivity of refractory cells to trastuzumab. Derivatives of NDGA are currently in clinical trial for several solid tumors.[59]

Poly(ADP-ribose) Polymerase Inhibitors

The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) represents an important novel target in cancer therapy. It is essential for the repair of single-strand DNA breaks via the base excision repair pathway. Preclinical data have suggested that single agent PARP inhibitors can selectively kill cancers with BRCA-1 or 2 mutations and cancers harboring defects in other DNA repair proteins.

Iniparib (BSI-201) is an intravenously administered PARP1 inhibitor that demonstrated significant antitumor activity in preclinical studies in vitro and in vivo when given either alone or in combination with chemotherapy. In recent randomized phase II clinical trial addition of iniparib to gemcitabine and carboplatin was found to improve CB rate, PFS, and OS in patients with metastatic TNBC compared with gemcitabine and carboplatin alone.[60] Other phase II/III clinical trials of iniparib as a single agent or in combination with chemotherapy are ongoing in other tumor types, such as ovarian and uterine cancer, NSCLC and glioblastoma.

Olaparib (AZD-2281), AG14361, NU1025, and 3-aminobenzamide are other novel, orally active poly(ADP-ribose) polymerase (PARP) inhibitor, that induced synthetic lethality in BRCA-deficient cells. Currently, more than a dozen clinical trials are testing PARP inhibitors as single-agent or in combination with chemotherapeutic agents in breast cancer.

Histone Deacetylase Inhibitors

Histone deacetylase inhibitors (HDACi) are a novel class of antitumor agents that inhibit proliferation and induce differentiation and/or apoptosis of tumor cells by interfering with the function of histone deacetylase. HDAC inhibitors have been associated with a transcriptional down-regulation of ER and its response genes in ER-positive cancer cells. HDACi also sensitizes cancer cells to topoisomerase inhibitors by increasing their access and binding to DNA. In a phase II trial, HDACi vorinostat with doxorubicin exerted antitumor activity in breast cancer, prostate cancer, and melanoma and HDAC2 expression was predictive of HDAC inhibition.[61] Valproic acid is another HDACi that was well tolerated with epirubicin and had noteworthy antitumor activity in heavily pretreated patients and historically anthracycline-resistant tumors.[62]

Other Agents

Other novel molecular targets for which small molecule inhibitors or Mabs have been developed include c-Met inhibitor (ARQ-197), proteosome inhibitors (Bortezomib, BU-32), Aurora kinase inhibitors (AZD1152, CYC116), Integrin-Linked Kinase Inhibitor (QLT0267) and RANKL inhibitor (denosumab) to name a few. These agents are currently under investigation in several preclinical and clinical settings and are anticipated to demonstrate promising activity against breast cancer.

Future Directions

In the past decade, breast cancer therapy has dramatically improved partly due to early detection methods (e.g., mammography) and partly due to targeted therapy. We have reviewed several promising small molecule inhibitors and monoclonal antibodies that are used in various stages of clinical development for the treatment of breast cancer. With the increasing understanding of the molecular biology of signaling pathways the future of breast cancer therapy looks promising. Despite great interest in targeted agents, several/either had only modest activity or failed to perform to the expectations when used as single agents in unselected patients. It is becoming clear with more clinical trials that unleashing the clinical potential of these targeted agents would require pre-selection of patients, better assays to guide dose, knowledge of mechanism-based side effects, and ways to predict and overcome drug resistance. On the other hand, selection of patients for targeted therapy has been challenging in several cases due to the lack of reliable biomarkers to predict the efficacy of the targeted agents. Hence, there is an urgent need to identify novel biomarkers that can be used in clinical settings as new targeted drugs are discovered.

Finally, neoadjuvant approach in less pretreated patients is required for these targeted agents to gain early indications of clinical response and also incorporate sequential sampling for biomarker studies.