Breast Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

Breast Cancer Signaling Pathway

Fig.1 Breast cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in green) and those in preclinical or early phase development (colored in red) for the treatment of advanced stage breast cancer.

An Introduction to Breast Cancer

Breast cancer (BC) is the leading cause of cancer death among women worldwide. The vast majority of breast cancers are carcinomas that originate from cells lining the milk-forming ducts of the mammary gland. As a heterogeneous disease, breast cancer encompasses a wide variety of pathological entities and this heterogeneity is reflected by the differences in cell type composition and proportions, the differences in the proliferation ability between glandular and myoepithelial cells, the proliferation of progenitor cells, the therapeutic responses and patient outcomes. The molecular subtypes of breast cancer, which are based on the presence or absence of hormone receptors (estrogen and progesterone subtypes) and human epidermal growth factor receptor-2 (HER2), include: hormone receptor positive and HER2 negative (luminal A subtype), hormone receptor positive and HER2 positive (luminal B subtype), hormone receptor negative and HER2 positive (HER2 positive), and hormone receptor negative and HER2 negative (basal-like or triple-negative breast cancers (TNBCs)). Hormone receptor positive breast cancers are largely driven by the estrogen/ER pathway. In HER2 positive breast tumours, HER2 activates the PI3K/AKT and the RAS/RAF/MAPK pathways, and stimulate cell growth, survival and differentiation. In patients suffering from TNBC, the deregulation of various signalling pathways (Notch and Wnt/beta-catenin), EGFR protein have been confirmed.

1 Main Signaling Pathways in Breast Cancer Therapy

1.1 RTK signaling cascade

The activation of the RTK cascade is mostly initiated at membrane receptors, such as HER2, EGFR, IGFR, FGFR1, KIT. The binding of receptors and ligands activate the phosphatidylinositol-3-kinase (PI3K) or Ras signaling pathway. When PI3K is activated, it phosphorylates inositol-containing membrane lipids. The phosphorylation product PIP3 binds to AKT. In addition, PTEN have no activity to inhibit Akt activity by dephosphorylation of PIP3 in breast cancer cells. AKT then phosphorylates several substrates, leading to the activation of the so-called mammalian target of rapamycin (mTOR). When Ras pathway is activated, Ras proteins transduce signals from extracellular growth factors by cycling between inactive GDP-bound and active GTP-bound states and turn on the downstream RAF protein kinases. The dominant substrates of RAF kinases are the MAPK/ERK kinases, MEK1 and MEK2. ERK generates extensive changes in gene expression mediated by transcription factors that control cell cycle progression, differentiation.

1.2 Notch signaling cascade

The activation of Notch cascade is mostly initiated at the binding of extracellular domain of Notch receptors (NICD) and ligands (Jagged, Delta). NICD translocates to the nucleus, causing transactivation of downstream target genes including several genes (Hes, Hey). These transcriptional targets include transcription factors (NF-κB2 and c-Myc), cell-cycle regulators (cyclin D1 and p21), and growth factor receptors (HER2) and regulators of angiogenesis and apoptosis. So the interruption of the Notch pathway can thus have noteworthy downstream effects on differentiation, angiogenesis and apoptosis, cell growth.

1.3 Wnt signaling cascade

The Wnt pathway is activated by the up regulated Wnt receptor frizzled-7 (FZD7) and the Wnt co-receptor LRP6. Signaling via Dishevelled and/or Axin then results in inactivation of a multiprotein complex that normally renders β-catenin unstable. Once in the nucleus, β-catenin binds to proteins of the T-cell factor (TCF)/lymphoid enhancer factor-1 (LEF1) family and modulates the expression of several target genes. Several Wnt/β-catenin target genes have been identified, including those that control cell proliferation and apoptosis, thus mediating cancer initiation and progression.

1.4 Estrogen signaling cascade

The Estrogen pathway of breast cancer through ‘nuclear-initiated steroid signaling’ mediates its cellular actions. The cytoplasmic estrogens bind directly to the nuclear membrane estrogen receptor which in turn translocates to the nucleus, binds DNA at ERE elements and activates the expression of ERE-dependent genes. These ERE-dependent genes appears to regulate a set of genes involved in the differentiation and proliferation of breast cancer cells.

2 Breast cancer diagnosis

2.1 Molecular Markers for Breast Cancer

Although the most of these markers is protein, gene expression patterns and altered DNA identified in tumor tissue have also taken prominence as tumor markers. It is known that breast cancer represents a complex and heterogeneous disease that comprises distinct pathologies, histological features, and clinical outcome. Also, it is well established that this neoplasia has well-defined molecular subgroups based on gene expression profiling closely related to the behavior of these molecular subtypes. Some researchers pointed out that results from studies of gene expression profiling have altered the view of breast cancer and provided a new tool for molecular diagnosis. Actually, the status of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor type 2 (HER2) has been used as predictive markers for identifying a high-risk phenotype and for selection of the most efficient therapies. After the sequencing of human genome and the technical progress in protein identification, it is reasonable considering an integrated program of genomics and proteomics to accomplish better comprehension of breast cancer features and the development of improved therapeutics. Together, these results strengthened evidence of improved sensitivity and resolution methodologies, which contribute to the classification of breast cancer.

RNA and DNA can also be tested in routine paraffin-embedded tissue samples, and in situ hybridization can detect HER2 amplification as a confirmatory test for IHC or as a stand-alone assay. There is great interest in other actionable targets in cancer genomes for precision therapy using next-generation gene sequencing. DNA microarrays and high-throughput reverse transcription-polymerase chain reaction assays for multiple genes can be used to categorize breast cancers into several prognostic groups. Gene assays are used to predict the risk of distant recurrence in early-stage breast cancer and to influence decisions about systemic therapy. These tests rely heavily on the assessment of ER and proliferation-related genes, such as Ki-67, and have largely replaced the use of other, single markers of risk in clinical practice.

2.2 Protein Markers for Breast Cancer

Women with the so-called ‘triple negative’ subtype of breast cancer have poor prognosis and survival compared to other subtypes due to the aggressive nature of these tumours and the current absence of suitable targets for therapy.

The majority of breast tumours are ER, PR, or HER2+ positive and can be effectively treated with targeted therapies directed against these proteins, such as hormonal therapies blocking production or function of oestrogens, and antibody or kinase inhibitor therapies blocking the HER2 pathway. However, about 15 percent of breast tumours are triple negative and effective treatment is not readily available. Identification of protein markers that are specifically expressed in triple negative tumours would not only benefit proper clinical classification, but would provide the basis for development of new, targeted therapies against this aggressive type of breast cancer. Critical treatment decisions are made on the basis of protein expression assays that are independent of tumor morphologic characteristics. IHC analysis of paraffin sections is routinely performed for the evaluation of estrogen receptor (ER), progesterone receptor (PR), and Her-2/neu (HER2) status. Although widely used to predict responses to targeted agents, histologic tumor markers are limited by significant intratumoral variation, even within a single biopsy specimen.

3 Targeted therapy for breast cancer

The RTK /Notch/Wnt/Estrogen signaling pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The aberrant expression of: HER2, ER, PR, BRCA1, BRCA2, EGFR1, PIP3, PTEN, P53, as well as other oncogenes and tumor suppressor genes. A greater understanding of the underlying biology of breast cancer has resulted in the identification of a number of molecular targets and development of  therapeutics intervention.

We have focused on recently discovered therapeutic targets for breast cancer, such as the RTK signaling pathway, Notch signaling pathway, Wnt/β-catenin pathway, Estrogen/ER signaling pathway, EGFR inhibitors, PARP1 inhibitors, and mammalian target of rapamycin (mTOR) inhibitors, as well as a few therapeutic options that have recently been developed for the treatment of breast cancer based on these targets along with their progress in clinical trials (Table 1-13).

3.1 Breast cancer therapy for BRCA1 and BRCA2 mutations

PARP1, a poly ADP-ribose polymerase, is a member of a family of enzymes that remove damaged and (or) incorrect DNA sequences by different excision repair pathways, involving base excision repair, nucleotide excision repair and mismatch repair by filling the ensuing gap using the complementary DNS strand as a template. Therefore, a cancer that involves a mutation in these BRAC1 and BRAC2 genes (BRCAness) can be effectively treated with PARP1 inhibitors such as Olaparib, Iniparib, which may represent very efficient therapies for breast cancer because of the high sensitivity of these tumours to the inhibitor and more importantly.

Table 1 Clinical trials of PARP1 inhibitor Olaparib

Nct id

Status

Lead sponsor

Study first posted

According to statistics, a total of 17 Olaparib projects targeting breast cancer BRCAness are currently in clinical stage, of which 14 are recruiting and 3 are not recruiting.

3.2 Breast cancer therapy for RTK pathway

The RTK pathway generates Akt (PKB) and MAPK causing drug resistance. In addition, it regulates cell proliferation, differentiation, invasion, angiogenesis and apoptosis through various signaling pathways and serves as a poor prognostic factor in cancer, and the RTK pathway could be a therapeutic target for women with BC that effectively treated with HER2 inhibitors Herceptin, Lapatinib, EGFP inhibitors Gefitinib, Cetuximab, KIT inhibitors Sorafenib, mTOR inhibitors Everolumus, Temsirolimus, Cisplatin, Erlotinib.

Table 2 Clinical trials of HER-2 mAB Herceptin

Nct id

Status

Lead sponsor

Study first posted

Table 4 Clinical trials of HER-2 inhibitors Lapatinib

According to statistics, a total of 117 Herceptin projects targeting breast cancer HER2 are currently in clinical stage, of which 78 are recruiting and 39 are not recruiting.

Table 3 Clinical trials of HER2 inhibitor Lapatinib

Nct id

status

Lead sponsor

study first posted

According to statistics, a total of 23 Lapatinib projects targeting breast cancer HER2 are currently in clinical stage, of which 8 are recruiting and 15 are not recruiting.

Table 4 Clinical trials of EGFR mAB Cetuximab

Nct id

Status

Lead sponsor

study first posted

Table 5 Clinical trials of KIT inhibitors Sorafenib

Nct id

Status

Lead sponsor

study first posted

Table 6 Clinical trials of mTOR inhibitors Cisplatin

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 25 Cisplatin projects targeting breast cancer mTOR are currently in clinical stage, of which 20 are recruiting and 5 are not recruiting.

Table 7 Clinical trials of mTOR inhibitors Erlotinib

Nct id

Status

Lead sponsor

study first posted

Table 8 Clinical trials of mTOR inhibitors Temsirolimus

According to statistics, a total of 37 Temsirolimus projects targeting breast cancer mTOR are currently in clinical stage, of which 21 are recruiting and 16 are not recruiting.

Table 8 Clinical trials of mTOR inhibitor Everolumus

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 38 Everolumus projects targeting breast cancer mTOR are currently in clinical stage, of which 22 are recruiting and 16 are not recruiting.

3.3 Breast cancer therapy for Notch pathway

Notch receptors and ligands may be inhibited by selective strategiessuch as g-secretase inhibitors RO4929097, GSIs show antitumor activity in several human cancer cell lines. Xenograft studies with glioblastoma and lung adenocarcinoma cell lines have shown that GSIs reduced both tumor growth and vasculature, induced growth arrest of T-ALL cells, and induced apoptosis in melanoma cell lines.

3.4 Breast cancer therapy for Wnt pathway

Several Wnt/β-catenin target genes have been identified, including those that control cell proliferation and apoptosis, thus mediating cancer initiation and progression. In addition to this, Salinomycin and Nigericin, act as inhibitors of the Wnt/βcatenin signaling pathway by inducing degradation of LRP6, and are selective breast cancer stem cell killers.

3.5 Breast cancer therapy for estrogen pathway

The estrogens first penetrate through the cell membrane and then bind to the estrogen receptor monomer, which is then dimerized and transported into the nucleus to activate signaling. Treatment with tamoxifen blocks the binding of estrogen and, thus, inhibits the estrogen-activated signaling. Treatment with Fulvestrant inhibits the cytoplasmic dimerization of estrogen. Treatment with aromatase inhibitors, such as Letrozole, Anastrozole and Exemestane blocks estrogen production. Another characteristic correlated to the ER pathway is the progesterone receptor (PR) signaling, which is also involved in the regulation of luminal type breast cancer development.

Table 9 Clinical trials of ER inhibitor Letrozole

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 77 Letrozole projects targeting breast cancer ER are currently in clinical stage, of which 45 are recruiting and 32 are not recruiting.

Table 10 Clinical trials of ER inhibitors Tamoxifen

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 75 Tamoxifen projects targeting breast cancer ER are currently in clinical stage, of which 37 are recruiting and 38 are not recruiting.

Table 11 Clinical trials of ER inhibitors Fulvestrant

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 69 Fulvestrant projects targeting breast cancer ER are currently in clinical stage, of which 43 are recruiting and 26 are not recruiting.

Table 12 Clinical trials of ER inhibitors Anastrozole

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 56 Anastrozole projects targeting breast cancer ER are currently in clinical stage, of which 31 are recruiting and 25 are not recruiting.

Table 13 Clinical trials of ER inhibitors Exemestane

Nct id

Status

Lead sponsor

study first posted

According to statistics, a total of 63 Exemestane projects targeting breast cancer ER are currently in clinical stage, of which 38 are recruiting and 25 are not recruiting.