The Molecular Biology of Breast Cancer

Published on 09/04/2015 by admin

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This table is not comprehensive, but provides an example of breast cancer multiplexed gene tests that are currently in development or have been commercialized. The type of sample, number of genes, indication, and ability to predict therapy may change based on commercial development. Prediction of response to therapy has been shown for many of the tests; however, for some of the tests this may not be used or approved as an indication for the test.
Chemo, Chemotherapy; ER, estrogen receptor; FDA, U.S. Food and Drug Administration; FFPE, formalin-fixed paraffin-embedded; IVDMIA, In Vitro Diagnostic Multivariate Index Assay; RT-PCR, reverse transcriptase polymerase chain reaction; TAM, tamoxifen.

Despite extensive analysis of DNA changes (copy number, somatic mutation, and structural change) in breast cancer, relatively few multianalyte tests are currently available.

Molecular Basis of Breast Cancer

ER Action

The steroid hormone estradiol signals through two related receptors, ERα and ERβ. 74 Data from in vitro studies and mouse models, as well as studies using clinical specimens, have provided concrete evidence that ERα is the dominant regulator of both normal breast development 3 and breast cancer. 75 ERα (referred to hereafter as ER) is a nuclear hormone receptor that binds the estradiol with very high affinity. Ligand binding changes receptor conformation to allow binding to enhancer elements in DNA. Initial studies suggested that these DNA elements were close to promoters and provided a simple model of how ER directly affects promoter activity. However, advances in genome-wide DNA binding (e.g., ChIP-seq), chromatin conformation (e.g., Chia-PET), and RNA transcription assays (e.g., GRO-seq) have shown that ER action is much more complicated than previously thought. 76,77 ER often binds hundreds of kilobases upstream of promoters to regulate transcription via looping of large segments of DNA. ER’s action is regulated through its interaction with numerous co-regulatory proteins, which can either activate or repress its transcriptional activity. 78 Several of those co-regulators have been shown to be associated with endocrine resistance, such as SRC1, 79,80 SRC3, 81,82 and NCoR1. 83
Although ER clearly functions as a classical ligand-dependent DNA-binding transcription factor, it may also function in an extranuclear nongenomic manner. 84 This is, at least in part, mediated via growth factor activated signaling pathway, ultimately leading to phosphorylation of ER, especially at S118, S167, and S305, and subsequent recruitment of co-regulators and DNA binding. 85 Although such a role is mechanistically attractive, especially with regard to ER being associated with metastatic processes through interaction with SRC, PI3K, and MAPK signaling, the clinical relevance of ER nongenomic action in breast cancer is controversial. A well-controlled large study using more than 3000 clinical specimens showed that cytoplasmic ER expression occurs at a very low incidence rate of less than 2%. 86 More recent genome-wide studies of ER action are revealing new insight into ligand-independent action. For example, ER ChIP-seq of breast cancer cells treated with EGF identifies an ER cistrome that mediates regulation of genes involved in endocrine resistance in HER2-overexpressing tumors 87 and has also demonstrated critical roles for the chromatin pioneer factor, FoxA1. 88
Molecular biology studies of the structure and function of ER have had a profound effect on the development and use of anti-ER therapies for breast cancer. The first anti-ER ligand, tamoxifen, was originally developed as a contraceptive but never proved useful. 89 However, several studies showed that tamoxifen was highly successful as a targeted antihormonal therapy for women with all stages of ER-positive breast cancer. 90 Tamoxifen binds the ER, but does not activate gene expression (and indeed it represses many genes). A crystal structure of the ER showed that estradiol binding alters the conformation of the protein to cause movement of helix 12 and allow coactivators to bind ER and increase transcription. 91 Tamoxifen, in contrast, does not cause this molecular switch, in part explaining its antagonistic activity. However, tamoxifen exhibits tissue-specific activity and can be an agonist in tissues such as the uterus and bone, leading to its identification as a selective estrogen receptor modulator (SERM) that exhibits mixed agonist/antagonist activity. 92 Unfortunately, tumors can often exploit the agonist activity of tamoxifen and thus reduce its clinical effectiveness. Many molecular mechanisms for the well-studied area of tamoxifen resistance have been deciphered, with the most studied being increased growth factor signaling. 75
Another approach to inhibiting ER action is to block production of estradiol via inhibition of the enzyme aromatase in postmenopausal women or suppression of ovarian steroid production (by luteinizing hormone releasing hormones or LHRH agonists) in premenopausal women. 93 The advantage of this method over tamoxifen is that neither aromatase inhibitors nor LHRH agonists show agonist activity. Indeed, aromatase inhibitors have been shown to be superior to tamoxifen for the treatment of early and advanced ER-positive disease in postmenopausal women. 93 Interestingly, however, the metabolism of steroid hormones is complex, and other metabolites may activate the ER to circumvent the loss of activity due to a reduction in estradiol. 94,95 A recent whole-genome sequencing of breast tumors before neoadjuvant aromatase inhibitor therapy has revealed mutations in primary breast cancer that map to several signal transduction pathways, and increased mutation of the TP53 pathway (38%) in aromatase inhibitor–resistant tumors compared to those that responded to therapy (17%).
Perhaps the most logical approach to blockade of ER action in breast cancer would be the total removal of ER protein such that no ligand-dependent or independent activation could occur. ICI 182780 (fulvestrant, AstraZeneca) is a selective estrogen receptor downregulator (SERD) that is similar in structure to estradiol, binds ER with the same affinity, and leads to rapid receptor degradation. 96 The actual mechanism of degradation is unknown but likely involves the proteasome. Clinical development of fulvestrant has been hampered by the fact that it requires regular intramuscular injection, and early trials likely used doses (250 mg) that were not sufficient to saturate ER. A recent Phase II trial comparing first-line fulvestrant (500 mg) to the aromatase inhibitor, anastrozole, showed superiority in time to tumor progression for fulvestrant. 97 Furthermore, anastrozole plus fulvestrant was recently reported to be superior to anastrozole alone. 98 Further delineation of the molecular mechanisms whereby ER activates gene transcription, and how SERMs and SERDs inhibit ER activity, will likely lead to the development of improved anti-ER therapies that minimize the emergence of therapeutic resistance.

Chromatin Remodeling

It is now commonly accepted that epigenetic changes such as DNA methylation, chromatin changes, and regulation of gene expression by miRNA play a role in carcinogenesis in many tumors, including breast cancer. 99 Aberrant DNA methylation has been studied extensively, both at the single gene and genome-wide levels. A number of genes have been reproducibly shown to be methylated in breast tumors, such as RASSF1A, PR, RARβ, CCND2, and BRCA1. However, at this point, no predictive or prognostic marker includes measurement of methylation. 100
Unexpectedly, sequencing studies of tumors have revealed very frequent somatic mutations in chromatin-modifying genes, including in the family of ATP-dependent chromatin remodeling proteins, enzymes modifying posttranslational modification of histones, and histone variants. For example, mutations in the H3K4 methyltransferase MLL are among the most frequent in breast tumors. 63,101 Other histone-modifying enzymes are highly expressed in aggressive breast tumors, such as the H3K27 methyltransferase EZH2, which was also shown to contribute to the expansion of progenitor cells. 102
It is therefore not surprising that there are many efforts to target deregulated epigenetic pathways in breast cancer. In contrast to hematopoietic malignancies, there are currently no approved breast cancer epigenetic therapies. However, trials are ongoing with drugs that inhibit HDACs and DNA methyltransferases as well as efforts to target other histone-modifying enzymes, such as EZH2. 99

Growth Factors

Growth factors play a major role in both mammary gland development and breast cancer and have been studied intensely as therapeutic targets. 103 The best studied growth factor receptor in breast cancer is HER2 (ErbB2). HER2 is amplified in approximately 20% of breast cancers, and its amplification and/or overexpression is associated with poor prognosis. 104 HER2 is a member of the larger HER/ErbB family consisting of epidermal growth factor receptor (EGFR/ErbB1/HER1), ErbB3/HER3, and ErbB4/HER4. Amplification of HER2 is thought to cause increased homo- and heterodimerization with other family members, resulting in constitutive activation of downstream signaling pathways leading to cancer cell growth and survival. The identification of this dominant activating oncogene led to one of the first examples of bedside-to-bench translational research with the development of monoclonal antibodies that block HER2. Trastuzumab (Herceptin, Genentech, South San Francisco, Calif) is a humanized monoclonal antibody that binds the extracellular domain of ErbB2. Trials of trastuzumab plus chemotherapy as first-line therapy in advanced breast cancer improved response rate, time to progression, and overall survival. 105 Similarly, adjuvant use of trastuzumab significantly improves disease-free and overall survival. 106
Despite major advances in the management of HER2-positive breast cancer with trastuzumab, de novo and acquired resistance is common. This has led to a number of alternative strategies to target ErbB2. 107 Pertuzumab (Perjeta, 2C4; Genentech, South San Francisco, Calif) is a monoclonal antibody that, like trastuzumab, binds the extracellular domain of ErbB2. However, it binds a different part of the domain that is critical for dimerization of ErbB2 to ErbB3. Preclinical and early clinical trials suggest that pertuzumab is active in trastuzumab-resistant breast cancers and can also enhance trastuzumab efficacy when given in combination. This was recently demonstrated in the Phase III CLEOPATRA trial in women with advanced HER2-positive breast cancer 108 and is under further study in the MARIANNE, NEOSPHERE, TRYPHAENA, and APHINITY trials.
Therapeutic drugs targeting the ErbB family tyrosine kinase domains have been developed. 109 Lapatinib (Tykerb, GlaxoSmithKline, London, UK) is a small-molecule reversible inhibitor of both ErbB1 and ErbB2 kinase domains and has been approved for the treatment of HER2+ metastatic breast cancer. Neratinib (HKI-272, Pfizer, New York, NY), in contrast to lapatinib, is an irreversible inhibitor of all ErbB kinase domains. Similar to pertuzumab and lapatinib, neratinib has documented activity in trastuzumab-resistant preclinical models and clinical trials and is currently in multiple trials to define its role in the treatment of HER2+ breast cancer.
An alternative and novel approach to strictly targeting HER2 activity is trastuzumab emtansine (T-DM1; Roche, South San Francisco, Calif), a conjugation of an anti-microtubule agent (maytansine) to trastuzumab. Importantly, a comparison of T-DM1 to trastuzumab/docetaxel for first-line treatment of metastatic breast cancer showed both improved response (response rate and investigator-reported progression-free survival) and reduced toxicity for T-DM1. 107
Growth factors are major regulators of mammary gland development, but they act via an intricate regulation by the steroid hormones estrogen and progesterone. 2 Intriguingly, normal steroid receptor positive mammary epithelial cells do not proliferate in response to steroid hormones, but they send a paracrine signal (most likely IGF and other growth factors) to neighboring cells that then proliferate. 110 This paracrine regulation is thought to be critical for the branching morphogenesis of the developing mammary gland. The intricate interaction between steroid hormones and growth factors is likely one of the first pathways to become dysregulated in tumorigenesis, as transcriptomic analysis of early premalignant lesions found elevation of both ER and growth factor (EGF and IGF) signaling. 111 Crosstalk between steroid hormones and growth factors is apparent not only in normal mammary development, but also in breast carcinogenesis. For the EGFR/ErbB2 pathway, increased hormone signaling is generally associated with reduced signaling. For example, there is a negative correlation between ErbB2 and ER levels, and ER is a repressor of ErbB2 levels via PAX2. 112 In contrast, for the IGF/insulin pathway, ER and PR are both positive regulators, with estrogen in particular upregulating ligand, receptor, and downstream signaling component expression. 113 Although IGF-IR and ER are highly correlated in breast tumors, and thus IGF-IR correlates with good prognosis, recent studies examining IGF-IR specifically in TNBC have shown that it correlates with poor outcome and may be a good therapeutic target. 114
Experimental evidence from breast cancer cell lines has suggested that a major mechanism of resistance to antihormonal therapy is via upregulation of growth factor receptor pathways. 75 However, until recently, results from clinical trials testing this hypothesis have been disappointing, with relatively little benefit from adding anti-EGFR or anti-IGFR therapies to antihormonal therapy. However, in one promising trial, targeting of a signaling molecule mTOR, which is downstream of both IGF-IR and EGFR, showed that the combination of an mTOR inhibitor (everolimus) and an aromatase inhibitor was superior to the aromatase inhibitor alone in the treatment of hormone-resistant advanced breast cancer in postmenopausal women. 115

Angiogenesis

Tumors are generally avascular when they first start to grow; however, as the tumor progresses and increases in size, the distance of cells to blood vessels and nutrients necessitates the new formation of blood vessels (angiogenesis). This angiogenic switch is seen as a critical barrier to tumor growth. 116 Preclinical research has identified many critical factors in the angiogenic switch, and blocking this switch with inhibitors of vascular endothelial growth factor (VEGF) has shown benefit in many preclinical models. Clinical testing of VEGF inhibitors (monoclonal antibodies and tyrosine kinase inhibitors) in addition to chemotherapy for women with advanced breast cancer has shown improvements in progression-free survival but little or no effect on overall survival. 117 Although it was believed that targeting the host blood supply would circumvent cancer cell intrinsic mechanisms of resistance, resistance to VEGF inhibitors is rapid and via multiple mechanisms. 118 Future use of angiogenesis inhibitors in breast cancer will likely require the identification of biomarkers of response to optimize clinical benefit.

Conclusion and Outlook

Investigating the molecular biology of breast cancer has given tremendous insight into the development and evolution of the disease and highlighted pathways for therapeutic intervention. However, as techniques for interrogating the molecular underpinnings of breast cancer have allowed deeper insight, it is clear that the levels of molecular alteration are much greater than previously anticipated. Indeed, although tumors clearly share certain features (such as ER+ and/or ErbB2+), no two tumors are the same, and the difference in their evolution and expansion provides great challenges for targeted therapies. It is anticipated that the next generation of research will likely tackle two main areas: the heterogeneity of molecular alterations in tumors and the clonal origin of breast cancer. Answers to these two questions are likely to have broad implications for the prevention and treatment of breast cancer.

Acknowledgments

This work was supported in part by research grants from the NIH/National Cancer Institute R01CA94118 (AVL), R01097213 (SO), the Breast Cancer Research Foundation (SO and NED), Susan G. Komen for the Cure (AVL), the Pennsylvania Department of Health (AVL and SO), and the Pennsylvania Breast Cancer Coalition (SO).
All references are available at Expert Consult.
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27. Rios

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