The 70-gene profile covers the six hallmarks of cancer
The six hallmarks of cancer describe the acquired characteristics of a cancer cell that collectively dictate malignant growth and thus reflect the potential of a tumor to metastasize.11
These six capabilities are shared by most (and perhaps all) types of human cancer. In breast cancer, the 70 MammaPrint genes are predictive of metastasis development, and we set out to ask whether the biological properties of these genes are correlated with the six hallmarks of cancer (as illustrated in ):
- Evading apoptosis
- Self-sufficiency in growth signals
- Insensitivity to anti-growth signals
- Limitless replicative potential
- Tissue invasion and metastasis
- Sustained angiogenesis
Figure 1. Depicted is how the genes in 70-gene tumor expression profile are involved in the six well-defined hallmarks of cancer, in tumor progression and metastasis related biological processes, as well as epithelial-mesenchymal transition. Adapted from Cell, (more ...)
The hallmark avoiding apoptosis confers resistance towards programmed cell death. The major converging point of diverse apoptotic signals that a tumor cell may receive are the mitochondria. Mitochondrial death signals are governed by the BCL2 family of proteins that release cytochrome C and, in turn, activates caspases.12
Tumor cells can resist apoptosis amongst others by altering expression level of BCL2 or caspases associated proteins. This mechanism is represented by two of the MammaPrint genes (BBC3 and EGLN1
Biological function of MammaPrint genes and cancer hallmarks. MammaPrint genes are involved in all tumor progression and metastasis-related biological processes, and cover the six well-defined hallmarks of cancer.
The hallmark self-sufficiency in growth signals, refers to tumor cells’ reduced dependence on exogenous growth stimulations by generation of their own growth signals. This can be achieved by manipulating the level of growth factors and their receptors or by mutation/altered expression of signal transduction molecules. This characteristic behavior of tumor cells is captured by six growth factor associated genes in the MammaPrint profile (ESM1, IGFBP5, FGF18, SCUBE2, TGFB3, WISP1;
). They represent the capability of tumor cells to manipulate different signaling pathways, such as the IGF-1 signaling pathway, FGF signaling pathway, cell cycle G1/S checkpoint regulation and Wnt/β-catenin signaling pathway. However, it should be emphasized that when various growth factors produced by tumor cells co-exist, the effect which results from their interplay within the microenvironment13
remains to be elucidated.
Equally important, is the hallmark labeled insensitivity to anti-growth signals. This defines the capability of tumor cells to disrupt responses to antiproliferative signaling. A well-studied example is the disruption of growth inhibiting effect of TGFß during tumorigenesis.13
This pathway is represented by the TGFB3 gene in the MammaPrint profile.
The three hallmarks, evading apoptosis, self-sufficiency in growth signals and insensitivity to anti-growth signals, all lead to growth and proliferation of tumor cells, regardless of the types of exogenous signals received from the tumor microenvironment.11
Although the biological processes by which normal cells acquire these three capabilities can be quite diverse, the biological features of proliferation and oncogenic transformation are shared among malignant tumor cells (see ). These shared characteristic behaviors are captured by 12 proliferation or oncogenic transformation-related genes (FLT1, HRASLS, STK32B, RASSF7, DCK, MELK, EXT1, GNAZ, EBF4, MTDH, PITRM1, QSCN6L1
; ). Because future metastatic dissemination of a primary tumor is directly associated with the aggressiveness of the primary tumor, it is perhaps not surprising that genes associated with these three hallmarks make up the largest part (21 genes) of the MammaPrint profile (BBC3, EGLN1, TGFB3, ESM1, IGFBP5, FGF18, SCUBE2, TGFB3, WISP1, FLT1, HRASLS, STK32B, RASSF7, DCK, MELK, EXT1, GNAZ, EBF4, MTDH, PITRM1, QSCN6L1
A fourth hallmark of the tumor cell is its limitless replicative potential. Tumor cells bypass two built-in checkpoints that limit the replicative potential of normal cells, the p53 and RB-dependent M1 senescence checkpoint and the telomerase-dependent M2 checkpoint.14
Fifteen MammaPrint genes are cell cycle genes, and can be assigned to this important feature of tumor cells (CCNE2, ECT2, CENPA, LIN9, KNTC2, MCM6, NUSAP1, ORC6L, TSPYL5, RUNDC1, PRC1, RFC4, RECQL5, CDCA7, DTL
; ). Interestingly, the protein-protein interaction network analysis also identified TP53
to be in the center of this network and confirms that the 70 genes are controlled by key tumorigenesis regulators (vide infra and ).
Figure 2. Protein-protein interaction network analyses indicate that the 70 genes form highly interconnected networks centered on known cancer-related transcription regulators such as TP53, RB1, MYC, JUN and CDKN2A (highlighted in orange). This network indicated (more ...)
The hallmark of tissue invasion and metastasis is a fifth critical step that involves local invasion of the tumor cells into surrounding tissue, escape from the primary tumor site, entry of metastatic tumor cells into the vasculature (intravasation), transportation and survival into the circulation, and arrest and exit of metastatic tumor cells from the vasculature into distant organs (extravasation).15
During the process of local invasion, tumor cells lose adhesion proteins, remodel extracellular matrix, gain motility by changes in their cytoskeleton and invade adjacent tissue.11
Five of the MammaPrint genes encode adhesion molecules, extracellular matrix constituents and proteins involved in the breakdown of extracellular matrix (COL4A2, GPR180, MMP9, GPR126 and RTN4RL1
; ). In addition to changes in cell adhesion and the extracellular matrix, tumor cells also have to acquire enhanced motility to successfully invade the surrounding tissue. A primary mechanism that regulates cell motility is the reorganization of the actin cytoskeleton.16
Actin-binding proteins regulate the dynamic assembly and disassembly of actin filaments that generate a protrusive force at the leading edge of the cell and a contractive force at the trailing edge of cell. These actin dynamics drive cellular motility. This specific malignant characteristic, enhanced motility, is addressed by three genes of the MammaPrint prognostic profile that relate to motility or actin filament organization (DIAPH3, CDC42BPA, PALM2
; ). In addition, four transformationrelated genes and growth factors are known to be critical for inducing local invasion,15
and are also represented in the MammaPrint 70 gene expression profile (TGFB3, IGFBP5, FGF18 and WISP1; ).
The last hallmark of cancer is sustained angiogenesis.17
The survival and growth of tumor cells depends on an adequate supply of oxygen and nutrients through blood vessels and by diffusion through the surrounding tissue. Existing vasculature and passive diffusion are sufficient for oxygen supply to tumors of a limited size. However, aggressive solid tumors often grow to a size that can no longer be sustained by the existing tissue vasculature. Tumor cells enhance their glycolytic capability to ensure an energetically efficient metabolism and proliferation rate under hypoxic conditions. These altered metabolic pathways are preserved by the tumor even when the oxygen concentration is sufficient.18
Seven genes implicated in this altered metabolism are represented in the MammaPrint profile (ALDH4 A1, AYTL2, OXCT1, PECI, GMPS, GSTM3
and SLC2 A3
; ). Tumor cells induce angiogenesis and vascular remodeling by regulating adhesion proteins and extracellular proteins through binding of hypoxia-inducible factor (HIF) to hypoxia-response elements (HRE).17
At least six genes in the MammaPrint profile are currently known to be direct effectors of angiogenesis and the regulation of vascular remodeling (FLT1, FGF18, COL4 A2, GPR180, EGLN1 and MMP9;
). Together, these genes assess the capability of tumor cells to stimulate the growth of new blood vessels and are likely to contain valuable information about the aggressiveness and malignant potential of a primary tumor.
It should be emphasized that the biological processes associated with the six hallmarks such as proliferation, cell-cell adhesion, angiogenesis and invasion are intrinsically linked. That is, a gene known to play a dominant and critical role in one hallmark might also indirectly be involved in other hallmarks. To better understand interactions between the 70 MammaPrint genes and their transcription regulation, we performed protein-protein interaction network analyses. The networks showed that the 70 genes are highly interconnected and center around known cancer-related transcription regulators such as TP53, RB1, MYC, JUN and CDKN2 A (). This result indicates that the activities of the 70 genes are regulated by these key tumorigenesis-related transcription regulators.
To summarize, MammaPrint has been developed using a data-driven approach and results in a gene profile that has comprehensive coverage of the six hallmarks of cancer, as well as tumor progression and metastasis related biological processes (, ). In addition, protein-protein interaction network analyses presented here, indicate that the 70-genes form highly interconnected networks and that their expression levels are regulated by key tumorigenesis related genes such as TP53, RB1, MYC, JUN and CDKN2 A.
The biological model of acquisition of metastatic competence through epithelial-mesenchymal transition and the 70-Gene Profile
In the previous section, we have shown that malignancy and metastatic competence of tumor cells at the primary tumor site are measured by the expression level of genes in the 70-gene MammaPrint profile. However, this has not provided an answer as to how tumor cells at the primary tumor site initially acquire their metastatic capability. A biological model that is increasingly gaining acceptance is that tumor cells at the primary site might acquire their metastatic capacity through a process similar to epithelelial-mesenchymal transition (EMT): a key epigenetic program that cells undergo during early embryonic development.8
During EMT, epithelial cells lose cell adhesion molecules, reorganize their cytoskeleton, gain increased motility and migrate from an epithelial sheet-like structure to an irregular structure of mesenchyme.19
This change in cellular phenotype is similar to the process that tumor cells undergo to initiate metastasis. Evidence suggests that tumor cells might initiate EMT by turning on or off some of the same transcription factors that are used in early embryonic development.20
These transcription factors regulate the expression of genes that allow tumor cells to lose adhesion, remodel the surrounding extracellular matrix, acquire enhanced motility to enable cellular migration, resist apoptotic signals, and adapt to an unfamiliar microenvironment at the distant site. The biological model based on the assumption that EMT processes are involved in breast cancer metastasis is consistent with the biological functions of the genes in the MammaPrint 70-gene profile identified here (). A substantial number (ie, 14 genes) of the 70 gene profile encode for proteins that are known to play an role in early embryonic development and are likely involved in EMT (MMP9, COL4 A2, FLT1, TGFB3, IGFBP5, FGF18, WISP1, GPR180, ESM1, SCUBE2, PITRM1, EXT1, EBF4, ECT2
; ). Within the EMT-associated MammaPrint genes, one gene (EBF4) encodes development-related transcription factors and three genes (TGFB3, FGF18, WISP1
) represent the well characterized EMT-mediating TGF-β, FGF and Wnt family proteins.9
It should be noted that in addition to the described 14 genes, other genes within the 70-gene profile might also be involved in early embryonic development. However, their role in early embryonic development has not yet been studied extensively. As outlined above, these 14 EMT-associated MammaPrint genes show a significant overlap with genes that confer the capability of tissue invasion, extracellular matrix remodeling and enhanced motility of tumor cells. These are among the defining characteristics of the EMT phenomenon.9