Posts Tagged ‘Breast’

Targeting of GLUT1 Glucose Transporter

Larry H. Bernstein, MD, FCAP, Curator



Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells

Arieh Riskin, M.D., M.H.A., and Yehudit Mond, M.Sc.
Rambam Maimonides Med J  Oct 2015; 6( 4): e003         http://www.rmmj.org.il/userimages/525/1/PublishFiles/531Article.pdf

Abbreviations: ECFP, enhanced cyan fluorescent protein; EGFP, enhanced GFP; GFP, green fluorescent protein; GM, growth medium; HMEC, human mammary epithelial cells; MEC, mammary epithelial cells; MMEC, mouse mammary epithelial cells; pECFP, plasmid vector ECFP; pEGFP, plasmid vector EGFP; PCR, polymerase chain reaction; SM, secretion medium. Citation: Riskin A, Mond Y. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells. Rambam Maimonides Med J 2015;6 (4):e0038. http://dx.doi.org:/10.5041/RMMJ.10223

Background: Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. Objective: To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture.

Methods:Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin.

Results: GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90–110 minutes.

Conclusions: Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

KEY WORDS: CIT3 mouse mammary epithelial cells, green fluorescent protein, GLUT1 glucose transporter, human mammary epithelial cells, prolactin


Biology of Milk Production and Transport Pathways of Milk Constituents Females of all mammalians bear mammary glands, and milk secretion and lactation is a characteristic feature of all mammalian species, which are the only organisms that produce copious glandular skin secretions to feed their young.1,2 Lactation is a highly complex and evolutionarily ancient strategy of all mammals, providing their offspring with a highly digestible, concentrated, nutritionally balanced diet, while allowing adult mammals to evolve a wide range of developmental and reproductive strategies and specialize on diets that could either be too difficult to capture or digest or would be insufficient to cover the high nutritional needs of their small rapidly growing offspring.1–3 Lactation helps mammalian mothers cope with unreliable food supplies, because lactating females can draw on their nutrient reserves for milk production, suggesting an evolutionary advantage for their dependent offspring, since milk intake promotes growth, fitness, and survival of the young.2,4,5 Beyond nourishment of the neonate, milk also helps establish immunological and endocrine competence in the offspring. Milk’s nutrient composition varies extensively across mammalian species, as a function of evolutionary history, maternal nutrient intake, duration of milk production, and stage of lactation.2,5 Milk is a complex mixture whose composition reflects different transport and secretion mechanisms within the mammary gland that aim to answer the different nutritional needs of mammalian neonates.6

The lactating mammary gland is composed of branching ducts ending in alveolar clusters where milk is produced. A single layer of polarized secretory epithelial cells forms the alveolar wall. The alveoli are surrounded by myoepithelial cells and are embedded in vascularized connective tissue stroma. While growth of the mammary gland and secretion of milk are stimulated by growth hormone, prolactin, adrenocortical steroids, estrogens, and progesterone, ejection of milk requires contraction of myoepithelial cells stimulated by oxytocin.6,7 The cytoplasm of the secretory alveolar epithelial cells is filled with numerous mitochondria, extensive rough endoplasmic reticulum network, well-developed Golgi apparatus, and secretory vesicles in the apical region of the cell adjacent to the alveolar lumen. The basal side of the alveolar epithelial cells lies on a basement membrane that separates them from the stroma and vascular system. In between, epithelial cells are connected to each other by an apical complex of tight junctions that inhibit direct paracellular exchange of substances between the vascular compartment and milk in the alveolar lumen. There are five pathways by which solutes, including proteins, lipids, ions, nutrients, and water, can be transported into the milk. Four are transcellular, involving transport across at least two membrane barriers, while the fifth is para-cellular and allows direct exchange of interstitial and milk components: (1) The exocytic pathway is for endogenously generated aqueous soluble substances, including lactose, oligosaccharides, the major milk proteins, citrate, phosphate, calcium, and other nutrients, which is similar to exocytic pathways in other cell types; (2) The transport pathway for milk lipids is unique to the mammary gland generating the milk fat globules; milk lipids, primarily triacylglycerides, are synthesized in the smooth endoplasmic reticulum in the basal region of the lactating alveolar cell—the newly synthesized lipid molecules are coated by protein membranes to form small storage structures called cytoplasmic lipid droplets that are transported to the apical plasma membrane, where they are secreted by a unique budding process as membrane-enveloped structures, i.e. the milk fat globules; (3) The trans-cytosis pathway transports macromolecules derived from the serum or stromal cells, including serum proteins (such as immunoglobulin, albumin, and transferrin), hormones (such as insulin, prolactin, and estrogen), and stromalderived substances (such as secretory immunoglobulin A, cytokines, and lipoprotein lipase); (4) Various membrane transport pathways transport ions and small molecules (such as glucose, amino acids, and water) across the basal and apical plasma membranes of the polarized alveolar epithelial cell; and (5) The para-cellular pathway provides a direct route for transfer of serum and interstitial substances into the milk. These transport pathways are regulated by the functional stage of the mammary gland and by hormones and growth factors.6

Studying mammary gland biology, transport pathways of milk constituents, and their influence on milk production in general, and specifically investigating glucose transport mechanisms in mammary epithelial cells (MEC) that could influence lactose synthesis and thus milk volume, is important and could have many clinical implications, including in breastfeeding support to mothers, in the dairy industry, and in oncology. Breast cancer tumor cells may utilize some of the MEC transport pathways to support metabolically their rapid uncontrolled growth.6,8

Glucose Transport Pathways in the Mammary Epithelial Cells Human milk is rich in lactose, which is the major osmotic constituent of human milk and thus the major determinant of milk volume.9 Lactose is a disaccharide composed of glucose and galactose. Lactose is found only in milk and is the primary carbohydrate in milk. The final step in the biosynthesis of lactose from UDP-galactose and glucose is catalyzed by lactose synthetase, a complex of - lactalbumin and the Golgi enzyme 1,4-galactosyltransferase.10 1,4-Galactosyltransferase is embedded in the inner surface of Golgi membranes. It is membrane-bound and directed towards the lumen of the cisternal space.10,11 Galactosyltransferase is found in most tissues and is involved in protein glycosylation. In mammals 1,4-galactosyltransferase has been recruited for a second biosynthetic function, the production of lactose. This function takes place exclusively in the lactating mammary gland. Galactosyltransferase has a relatively poor affinity for glucose (Km~1 M). The affinity of this enzyme is profoundly modified by transient association with -lactalbumin, which creates a binding site for glucose, so that the affinity of the transferase for glucose increases about 500-fold (Km~2 mM).10 This allows the synthesis of lactose in the Golgi to occur at the physiological concentrations found in the mammary cell. -Lactalbumin is a milk whey protein that is not catalytically active by itself, but is necessary for the synthesis of lactose. The initiation of -lactalbumin synthesis that occurs at parturition is required for the initiation of copious milk production, but is neither the only factor nor the limiting factor controlling lactose synthesis.11,12 Availability of glucose and UDP-galactose to the lactose synthase enzyme complex in the Golgi apparatus may be rate-limiting for lactose synthesis. Many cells possess active mechanisms for the uptake of nucleotide sugars such as UDP-galactose into the Golgi, which are essential to protein glycosylation. In contrast, the mammary gland is unique in its need to transport free glucose into the Golgi. The main known isoform of glucose transporters expressed in the mammary gland is GLUT1 (SLC2A1).9,13,14–20 Levels of GLUT1 increase progressively during pregnancy, reaching their highest levels during lactation,15,16 and this is dependent on prolactin.21 In most cells GLUT1 normally resides in the plasma membrane and is responsible for basal glucose uptake. In polarized epithelial cells, including mammary cells, GLUT1 is targeted primarily to the basolateral membrane.22 The role of GLUT1 in glucose transport into Golgi has been controversial.15,23,24 Evidence from in vivo and in vitro studies demonstrated unique hormonally regulated intracellular targeting of GLUT1 from the plasma membrane to a low-density intracellular compartment in mouse mammary gland during lactation.16,23–25 Further work distinguished this compartment from Golgi, suggesting that the hormonally induced intracellular targeting of GLUT1 in lactating MEC is into a Brefeldin A-sensitive low-density vesicle that may represent a subcompartment of cisGolgi.25 This hormonally regulated subcellular targeting of GLUT1 may have an important role for lactose synthesis in MEC during lactation.

The aim of this work was to study GLUT1 subcellular targeting in living human and mouse mammary epithelial cells (HMEC and MMEC) in culture; and to study GLUT1 intracellular trafficking in living MMEC in culture under the effects of the lactogenic hormones that regulate it. Our hypothesis was that there would be dynamic basal trafficking of GLUT1 in living MEC that would target most GLUT1 to the basolateral membrane under maintenance conditions, and intracellularly upon exposure to prolactin, which should be the main hormonal stimulus that drives this translocation during lactation. Our studies could then complete the previous in vitro findings in fixed cell25 by adding the dynamic observations in living MEC that could in turn relate to the in vivo findings.16

GLUT1 Subcellular Targeting in HMEC Immunocytochemistry of HMEC in maintenance MEGM medium using highly specific anti-GLUT1 antibody demonstrated plasma membrane distribution of GLUT1 as well as an intracellular, mostly perinuclear, pattern (Figure 1A). After exposure to the prolactin-rich MESM for 4 days, GLUT1 was specifically targeted intracellularly, demonstrating a perinuclear pattern. A distinct nuclear membrane distribution of GLUT1 was also observed under these conditions (Figure 1B). In secretion medium GLUT1 green signal colocalized with the blue signal of pECFP-Golgi (Figure 2A, B, C). It also co-localized with the red signal of alpha-lactalbumin and alpha-mannosidase II (Figure 2D, E, F, and G, H, I, respectively). Partial co-localization was demonstrated with the red signal of beta-COP and with transferrin-Texas Red (Figure 3A, B, C, and D, E, F, respectively). No co-localization was demonstrated after staining the cells with the red stain, BODIPY-TR ceramide (Figure 4A, B, C, D, E, F). GLUT1 Fusion Chimeras to EGFP Exhibit Normal GLUT1 Targeting in Vitro We subcloned GLUT1 cDNA into pEGFP to create N- and C-terminus fusions. The recombinant plasmid vectors were introduced into CIT3 cells by transient liposome-mediated transfection, achieving fluorescent expression in 20%–30% of the cells.

Figure 1. Exposure to Prolactin Causes Intracellular Targeting of GLUT1. Cells were fixed and exposed to specific anti-GLUT1 primary antibody. Bar 15 m. A: In maintenance medium, GLUT1 demonstrates primarily a plasma membrane distribution as well as some intracellular mostly perinuclear staining. B: After exposure to prolactin-rich medium for 4 days, GLUT1 was specifically targeted intracellularly, demonstrating a perinuclear pattern, as well as a distinct nuclear membrane staining.

Figure 2. After Exposure to Prolactin, GLUT1 Colocalizes with ECFP-Golgi, alpha-Lactalbumin and alpha- Mannosidase II. Fluorescent images were captured 60 hours after transfection with 1 microgram of pECFP-Golgi. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1, anti-alpha- lactalbumin or anti-alpha-mannosidase II. GLUT1 is shown in green, and alpha-lactalbumin or alpha-mannosidase II in red after staining with FITC-conjugated and Texas Redconjugated secondary antibodies, respectively. ECFPGolgi emits cyan-blue fluorescence when exposed to fluorescent light at the appropriate wavelength. Bar 10 microm. A, D, G: GLUT1 signal. B: ECFP-Golgi signal. E, H: alpha-Lactalbumin and alpha-mannosidase II signals, respectively. C, F, I: Superimposed images. Perinuclear colocalization of GLUT1 and ECFP-Golgi is shown as areas of coincident staining (C). Co-localization of GLUT1 and alpha-lactalbumin or alpha-mannosidase II appear as areas of coincident staining, giving rise to yellow signal (F, I).

Figure 3. After Exposure to Prolactin, GLUT1 only Partially Co-localizes with beta-COP and TransferrinTexas Red in Endosomes. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1 or anti-beta-COP primary antibodies. Some cells were exposed shortly to transferrin-Texas Red staining before fixation and exposure to anti-GLUT1. GLUT1 is shown in green, and beta-COP in red after staining with FITC-conjugated and Texas Redconjugated secondary antibodies, respectively. Transferrin stain appears in red. A, D: GLUT1 signal. B, E: beta-COP and transferrin (short-term exposure) signals, respectively. C, F: Superimposed images. Partial co-localization of GLUT1 with beta-COP or transferrin in endosomes appears as areas of coincident staining, giving rise to yellow signal.

Figure 4. After Exposure to Prolactin, GLUT1 Does not Co-localize with BODIPY-TR Ceramide. Cells were maintained in prolactin-rich medium for 4 days, before they were fixed and stained with specific anti-GLUT1 primary antibody, or stained with BODIPYTR ceramide. GLUT1 is shown in green after staining with FITC-conjugated secondary antibody. BODIPY-TR ceramide appears in red. A, D: GLUT1 signal. B, E: BODIPY-TR ceramide signal. C, F: Superimposed image. There is little overlap of GLUT1 green signal with BODIPY-TR ceramide.

GLUT1 Fusion Chimeras to EGFP Exhibit Normal GLUT1 Targeting in Vitro We subcloned GLUT1 cDNA into pEGFP to create N- and C-terminus fusions. The recombinant plasmid vectors were introduced into CIT3 cells by transient liposome-mediated transfection, achieving fluorescent expression in 20%–30% of the cells.

Immunocytochemistry studies with antibodies against the C-terminus of native GLUT1 showed that the fluorescent signal of both GLUT1 chimeras to GFP co-localized extensively with native GLUT1 (Figure 5). This result validated the use of the GLUT1-GFP fusion proteins to study dynamic aspects of GLUT1 targeting.

Figure 5. GLUT1 Chimeras to GFP Co-localize with Native GLUT1 in CIT3 Cells in SM. EGFP-GLUT1 fusion protein (B) exhibits the same intracellular distribution as native GLUT1 (A). Superimposed images (C) demonstrate that co-localization of native GLUT1 and EGFP-GLUT1 fusion protein appears as areas of coincident staining, giving rise to yellow signal. GLUT1-EGFP fusion protein (E) exhibits the same intracellular distribution as native GLUT1 (D). Superimposed images (F) demonstrate that co-localization of native GLUT1 and GLUT1-EGFP fusion protein appears as areas of coincident staining, giving rise to yellow signal.

EGFP chimeras to GLUT1 demonstrated change in subcellular distribution, after 96 hours of exposure to SM. In GM the fusion proteins were targeted mainly to the basolateral plasma membrane. In SM GLUT1-EGFP chimeras were mostly targeted into the cell, exhibiting unique perinuclear distribution with punctate pattern scattered through the cytoplasm (Figures 5 and 6). Both the N- and C-terminus fusions to GLUT1 (EGFP-GLUT1 and GLUT1-EGFP, respectively) exhibited the same targeting patterns (Figure 6).

Figure 6. CIT3 Cells Expressing EGFP Fusion to the N- and C-termini of GLUT1 in GM and after 4 Days in SM. High-power images. Upper left: Plasma membrane targeting of EGFP-GLUT1 in GM. Lower left: Intracellular pattern of EGFP-GLUT1 signal in differentiated cells in SM. Upper right: Plasma membrane targeting of GLUT1-EGFP in GM. Lower right: Intracellular targeting of GLUT1-EGFP with perinuclear punctate distribution in cells exposed to SM. All images captured with 0.25 s exposure time and are at 40× magnification, except for the upper left image, which was optimized at 0.5 s exposure time and higher power (60×).

Degree of Differentiation Affects Intracellular Targeting of GLUT1 Static images of cells at different levels of differentiation in SM demonstrated that the degree of differentiation affected the level and distribution of GLUT1 targeting in perinuclear localization (Figure 7). In GM the GLUT1-EGFP signal was mostly targeted to the plasma membrane (Figure 7A). In SM GLUT1-EGFP fusion proteins exhibited the unique perinuclear punctate pattern, described above (Figure 7B). However, in the more differentiated MEC in SM, where vesicles and fat globules were morphologically prominent, the GLUT1-EGFP signal was no longer punctate, but targeted to the boundaries of these vesicles, still maintaining mostly perinuclear distribution. There was signal throughout the cytoplasm as well (Figure 7C, D). This differentiation-related GLUT1 subcellular targeting may also support the assumption that dynamic transport systems are involved in the trafficking of the GFP-GLUT1 fusion proteins, as will be shown below.

Figure 7. Degree of Differentiation Affects Intracellular Targeting of GLUT1. Static images of CIT3 cells in GM and at different levels of differentiation in SM. Panel A: In GM; B, C, D: In SM at different levels of differentiation. The upper left figure of each panel is GLUT1-EGFP signal. The upper right figure is staining of the same cells with BODIPY-TR ceramide (Molecular Probes, Inc., Eugene, OR, USA), which is a dye that marks the trans-Golgi in red. Living CIT3 cells were pre-incubated with 5 nmol/mL of BODIPY-TR ceramide at 4C for 30 minutes. Lower left figure is superimposed image of the upper figures. Lower right figure is phase contrast image of the cells to show the different levels of differentiation.

GLUT1 Intracellular Trafficking in Secretion Media Seems to be Dynamic Living CIT3 cells transfected with GLUT1-EGFP and kept in SM were followed by time-lapse imaging, where trafficking of GLUT1 fusion proteins could be seen (Figure 8; also available online as a supplemental YouTube video clip). This trafficking seems to be dynamic and could suggest that transport systems are possibly involved in it.

Figure 8. GLUT1 Intracellular Trafficking in SM is Dynamic. Figure 8 is also available online as a YouTube video clip at: https://youtu.be/lEby2cqSDek. The figure plates are from frames 8 minutes apart. All images captured with 0.25 s exposure time and are at 40× magnification.

Secretion Medium Induces GLUT1 Intracellular Targeting in Living Mammary Cells When living CIT3 cells, transfected with GLUT1- EGFP and kept in GM, were exposed to SM, dynamic trafficking of GLUT1 fusion proteins was demonstrated intracellularly, starting after approximately 50–60 minutes, with maximal intracellular targeting within 90–110 minutes. When the cells were returned to GM, most of the changes were reversible within 1–2 hours, although not fully, with redistribution of the fluorescent GLUT1 chimera mostly in the plasma membrane (Figure 9; also available online as a supplemental YouTube video clip).

Figure 9. Lactogenic Hormones in SM Induce GLUT1 Intracellular Targeting in Living Mammary Cells. Figure 9 is also available online as a YouTube video clip at: https://youtu.be/3oYv21jcAj4. The figure plates are from frames 16 minutes apart. All images captured with 0.5 s exposure time and are at 40× magnification.

Prolactin Induces GLUT1 Intracellular Targeting in Living Mammary Cells Exposure of CIT3 cells kept in GM to SM containing prolactin without hydrocortisone caused the same changes in GLUT1 subcellular targeting as were seen with full SM. Dynamic trafficking of GLUT1 fusion proteins intracellularly was demonstrated, starting after approximately 50–60 minutes, with maximal intracellular targeting within 90–110 minutes. When the cells were returned to GM, most of the changes were reversible within 1–2 hours, although not fully. The same response was reproduced with different prolactin concentrations (300 ng/mL, 30 ng/mL), as low as 3 ng/mL (compared to the 3 microg/mL of prolactin usually used in SM). Representative results after exposure of CIT3 cells kept in GM to 30 ng/mL prolactin are shown (Figure 10; also available online as a supplemental YouTube video clip). We were not able to demonstrate doseresponse relations with the different prolactin concentrations, possibly because the results are qualitative, rather than quantitative. There was no difference in the time required to achieve maximal effect with different prolactin concentrations. Secretion medium containing hydrocortisone (3 microg/mL as in full SM) without any prolactin caused no change in GLUT1 subcellular distribution.

Figure 10. Prolactin Induces GLUT1 Intracellular Targeting in Living Mammary Cells. The response here was reproduced with prolactin concentrations of 30 ng/mL. Figure 10 is also available online as a YouTube video clip at: https://youtu.be/bUkUHZ0soPI. The figure plates are from frames 20 minutes apart. All images captured with 2 s exposure time and are at 60× magnification.

DISCUSSION Human milk contains about three times more lactose than does rodent milk, suggesting that glucose transport mechanisms can be very important in humans. Thus, before studying GLUT1 subcellular trafficking in a CIT3 MMEC model, we first examined whether hormonally regulated subcellular targeting of GLUT1 occurs in HMEC upon conditions mimicking lactation. For this, we utilized immunofluorescent staining of HMEC to demonstrate prolactin-dependent co-localization of GLUT1 with several Golgi markers.

The distribution of GLUT1, as demonstrated using immunocytochemistry with C-terminusspecific anti-GLUT1 antibody, was primarily in plasma membrane, as well as some intracellular perinuclear punctate pattern, when the cells were maintained in baseline growth medium. Upon exposure to prolactin in secretion media, GLUT1 specifically redistributed from the plasma membrane, demonstrating a perinuclear distribution with a pattern that may represent the Golgi or related structures in the central intracellular vacuolar trafficking system. A distinct perinuclear membrane distribution of GLUT1 was also observed under these conditions. Co-localization studies gave insight as to the possible Golgi subcompartment that GLUT1 resides in. GLUT1 was targeted intracellularly, co-localizing with components of lactose synthetase complex. It co-localized with ECFP-Golgi, the cyan fluorescent protein fused to the membrane-anchoring signal specific to beta 1,4-galactosyltransferase, identifying the medial/trans region of the Golgi. This colocalization gave another spatial support to its role in the transport of free glucose for lactose synthesis by beta 1,4-galactosyltransferase in the inner cisternae of the Golgi apparatus. This was further supported by the co-localization of GLUT1 with alpha-lactalbumin, which is the milk whey protein that is not catalytically active, by itself, but which in association with beta 1,4-galactosyltransferase is necessary for the synthesis of lactose.53 The initiation of alpha-lactalbumin synthesis that occurs at parturition is required for the initiation of copious milk production, but is neither the only factor nor the limiting factor controlling lactose synthesis.54 GLUT1 also colocalized with the medial-Golgi marker, alpha- mannosidase II; however, it did not co-localize with the trans-Golgi marker, BODIPY-TR ceramide. Only partial co-localization was demonstrated with beta- COP, which is a cis-Golgi marker.

The co-localization studies suggest intracellular targeting to the central vesicular transport system, which may represent a cis/medial-Golgi subcompartment. These findings are in agreement with previous findings in the mouse mammary gland16 and in CIT3 MMEC.25,55 Partial co-localization was also noted with transferrin-Texas Red after brief exposure, which marks the endosomes. The possible identification of GLUT1 in endosomes suggests that GLUT1 sorting is a continuous, dynamic process.55 Further work delineating the molecular mechanism of GLUT1 sorting and the targeting determinants it recognizes should improve our understanding of a key regulatory step of milk production in the nursing mother.

Apparent nuclear membrane staining for GLUT1 seen in HMEC has not been reported previously in any cell type, and its significance is a matter for speculation and further study. There is evidence to suggest that the apical nuclear envelope may serve as an intermediary connection between the endoplasmic reticulum and the Golgi.56 Also, the perinuclear staining of GLUT1 noted may actually be one of the perinuclear endosomal recycling compartments.52

Our findings in HMEC illustrate a potential mechanism for the delivery of free glucose to the Golgi, in what may be the rate-limiting step for lactose synthesis and milk production. In addition to its widely recognized role in the uptake of glucose by cells, GLUT1 may also mediate glucose transport between intracellular compartments.

The in vitro study of a dynamic process required developing a system of living cells with labeling of GLUT1. Green fluorescent protein is a reporter molecule for monitoring gene expression and protein localization in vivo, in situ, and in real time.57–63 Green fluorescent protein is expressed in eukaryotic cells as a fusion protein that serves as a “fluorescent tag.” The use of fluorescent fusion proteins of GLUT1 allows the study of the same cells over time, permitting studies of exocytosis and endocytosis, not just steady-state distributions. Using GFP fusion to GLUT1 is ideal for studying intracellular trafficking and subcellular targeting of GLUT1 in MEC under hormonal stimulation. It also permits evaluation of chimeric protein targeting in an antibody-independent fashion and confirms that we are studying GLUT1 and not a novel, lactation specific glucose transporter isoform that shares the GLUT1 epitope. The GLUT1 cDNA sequence29 was subcloned into pEGFP and introduced into CIT3 cells by transient transfection, with over-expression of the fluorescent GLUT1 in approximately a quarter of the cells. The intracellular targeting of GLUT1- EGFP chimera was consistent from cell to cell.

Lactogenic hormones in SM changed subcellular targeting of GFP fusion chimeras to GLUT1 from a plasma membrane distribution to an intracellular pattern, predominantly perinuclear and punctate, but also throughout the cytoplasm. The fact that this pattern was consistent with the distribution of native GLUT1 supported the use of GLUT1 chimeras to GFP as a model for studying GLUT1 intracellular targeting in MEC. Since the behavior and intracellular distribution of both the N- and C-fusion chimeras of GLUT1 to GFP were consistently the same, further studies were carried out with only one of them (GLUT1-EGFP). The level of differentiation of the lactating MEC in SM affected the degree of GLUT1 intracellular targeting and the distribution of its cytoplasmic, mostly perinuclear, localization. This fits well the previous in vivo and in vitro findings16,25 and actually also points to the possible involvement of intracellular membrane vesicular trafficking systems in GLUT1 intracellular targeting.

Living mouse MEC kept in SM demonstrated dynamic trafficking of GLUT1-EGFP fusion proteins. Careful tracking of these fluorescent GLUT1 vesicles excluded random movement and actually suggested that the dynamic intracellular targeting of GLUT1 may be mediated through altering GLUT1 exocytosis and endocytosis.

When CIT3 cells kept in GM were exposed to SM, the changes in GLUT1 targeting from mostly a plasma membrane pattern to an intracellular pattern occurred within 60–120 minutes. The maximal intracellular translocation of GLUT1-EGFP green fluorescent signal after exposure to SM was noted at 100–110 minutes. Some of this effect was reversible within 60–120 minutes upon withdrawal of SM, but we were not able to demonstrate full reversibility of the process in our in vitro system. The relatively rapid changes in GLUT1 targeting in living MMEC exposed to SM, which took place within minutes to hours, were in accordance with the findings from the in vivo studies of forced weaning, demonstrating reversible changes in GLUT1 subcellular targeting within 3–5 hours.16 These findings are also supported by the previous observation that, as early as 15 minutes after exposure of mammary tissue fragments from lactating rabbits to prolactin, the cell morphology already changed with marked increase in the relative volume occupied by the Golgi region.64

The next step was to define which of the hormones in SM is responsible for GLUT1 intracellular targeting. Exposure of CIT3 cells kept in GM to SM containing prolactin without hydrocortisone caused the same changes in GLUT1 subcellular targeting as seen with full SM. The same response was reproduced with prolactin concentrations as low as 3 ng/mL (compared to the 3 g/mL of prolactin usually used in SM). The serum concentration range of prolactin in lactating mothers is 20–300 ng/mL.65 However, we were not able to demonstrate dose-response effects with the different prolactin concentrations. The GLUT1-EGFP intracellular signal translocation took place at approximately the same time (100–120 minutes) with 3 ng/mL prolactin as it did with 3 g/mL of prolactin in the full SM. Further studies are needed to demonstrate dosedependent effects of prolactin, expressed as different levels of intracellular green fluorescent signal of GLUT1 chimeras, but this requires a more quantitative recording of the signal that unfortunately we did not have in these studies. Secretion medium containing hydrocortisone without any prolactin caused no change in GLUT1 subcellular distribution, thus excluding it as a cause for GLUT1 intracellular targeting in SM. Further studies are also needed to explore the effects of prolactin combined with other hormones, such as estrogen.

The suggestion that GLUT1 does not solely act at the plasma membrane, but may function in an intracellular organelle as well, conceptually complements the well-known insulin-regulated targeting of GLUT4,66 and to a lesser extent of GLUT1,38 to their site of action, the plasma membrane, in fat and muscle cells. Our results suggest the existence of a prolactin-induced, cell type-specific, developmental stage-specific sorting machinery for GLUT1 in MEC, and supports glucose transport as a potential ratelimiting step for lactose synthesis during lactation. The ability of the system to respond quickly to hormonal changes by altering the transport, and thus the availability of free glucose for lactose synthesis, is complementary to the well-known insulin-regulated targeting of GLUT4 to the plasma membrane in fat and muscle cells, where GLUT4 is available for glucose uptake into the cell within minutes.67 This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours, as was demonstrated also in vivo. 16 This step may not require new protein synthesis, or increase in the total amount of GLUT1 or enzymes involved in lactose synthesis, which takes longer. Our study relied only on immuno-histochemistry analysis, and further studies including Western blot and real-time PCR are needed to address the possibility of up- or downregulation of GLUT1.

Despite the fact that in our first experiments we have demonstrated specific intracellular targeting of GLUT1 from the plasma membrane of HMEC upon exposure to prolactin, the main limitation of this part of the dynamic studies is that it was limited to MMEC, and more specifically to CIT3 cell lines. Thus, these conclusions cannot be currently generalized or extended to other mammals, including humans. The suggestion that glucose transporters, other than GLUT1, may be involved in glucose regulation in MEC during lactation,18,68–71 and that their role may be more significant in other mammals, and influenced by factors other than lactogenic hormones,72–75 needs to be addressed.

Another limitation of this study is the issue of cell-to-cell variability in the phenotype of mouse MEC in the culture. Cell phenotype could vary in many ways, including morphology, intracellular lipid deposition, apparent states of differentiation, and GLUT1 targeting kinetics. This is an inherent limitation of a descriptive study based on microscopic findings. To decrease a possible selection bias of the cells we were studying, we tried to select representative cells on a lower-power field before studying them in a high-power field. We also repeated each experiment more than three times, and verified that the results were reproducible. Yet, such selection bias cannot be fully excluded.

A further limitation of our descriptive colocalization studies is the use of fluorescent microscopy. Confocal microscopy should be preferred in future studies like this, because it gives higher resolution and better color separation, enables the use of more colors at the same time, and is more accurate in differentiating true colocalization from proteins in close proximity.

Also, being a descriptive morphological study, our study did not deal with the ability of the cells to synthesize and secrete lactose in culture. This is a complex issue that is not dependent only on the presence of lactogenic hormones in the medium, and may be affected by many factors, such as the intracellular matrix. However, if these results were obtained in cells expressing lactose, then the issues of cell-to-cell variations and possible phenotypic effects discussed above would have been minimized.

The fact that this work is based on two previous works, one in vivo on mammary glands from lactating mice16 and the second in vitro on the same CIT3 cells, 25,55 establishes a continuum that supports our results within their limited scope. These results can possibly form the basis and methodological approach for future works in other primary MEC in order to try and generalize the conclusions.

In summary, our results demonstrated a basal constitutive GLUT1 membrane-recycling pathway between an intracellular pool and the cell surface in CIT3 MMEC, which targets most of the GLUT1 to the plasma membrane in GM. As in other cell types it is responsible for maintaining basal glucose uptake. But, in these MEC there is hormonally regulated cell type-specific, developmental stage-specific sorting machinery for GLUT1 intracellular targeting in lactation. This process is induced by prolactin and is highly sensitive to low concentrations of prolactin. It provides the cell with a quick mechanism by which it can supply free glucose intracellularly to serve as substrate for lactose synthesis in the Golgi. This machinery offers another level of regulation of lactose synthesis by altering GLUT1 targeting within minutes to hours, as was demonstrated also in vivo.16 The rapid responsiveness of GLUT1 targeting suggests that this machinery does not require new protein synthesis. It may also support glucose transport as a rate-limiting step for lactose synthesis during lactation.


1. Oftedal OT. The evolution of milk secretion and its ancient origins. Animal 2012;6:355–68. Full Text

2. Capuco AV, Akers RM. The origin and evolution of lactation. J Biol 2009;8:37. Full Text

3. Lefevre CM, Sharp JA, Nicholas KR. Evolution of lactation: ancient origin and extreme adaptations of the lactation system. Annu Rev Genomics Hum Genet 2010;11:219–38. Full Text

4. Dall SR, Boyd IL. Evolution of mammals: lactation helps mothers to cope with unreliable food supplies. Proc Biol Sci 2004;271:2049–57. Full Text

5. Skibiel AL, Downing LM, Orr TJ, Hood WR. The evolution of the nutrient composition of mammalian milks. J Anim Ecol 2013;82:1254–64. Full Text

6. McManaman JL, Neville MC. Mammary physiology and milk secretion. Adv Drug Deliv Rev 2003;55: 629–41. Full Text

7. Svennersten-Sjaunja K, Olsson K. Endocrinology of milk production. Domest Anim Endocrinol 2005;29: 241–58. Full Text

8. Strange R, Metcalfe T, Thackray L, Dang M. Apoptosis in normal and neoplastic mammary gland development. Microsc Res Tech 200115;52:171–81.

9. Zhao FQ. Biology of glucose transport in the mammary gland. J Mammary Gland Biol Neoplasia 2014;19:3–17. Full Text

10. Strous GJ. Golgi and secreted galactosyltransferase. CRC Crit Rev Biochem 1986;21:119–51. Full Text

11. Dils RR. Synthetic and secretory processes of lactation. Proc Nutr Soc 1989;48:9–15. Full Text

12. Nicholas KR, Hartmann PE, McDonald BL. AlphaLactalbumin and lactose concentrations in rat milk during lactation. Biochem J 1981;194:149–54. Full Text

13. Joost HG, Thorens B. The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review). Mol Membr Biol 2001;18: 247–56. Full Text

14. Burnol AF, Leturque A, Loizeau M, Postic C, Girard J. Glucose transporter expression in rat mammary gland. Biochem J 1990;270:277–9. Full Text

15. Camps M, Vilaro S, Testar X, Palacin M, Zorzano A. High and polarized expression of GLUT1 glucose transporters in epithelial cells from mammary gland: acute down-regulation of GLUT1 carriers by weaning. Endocrinology 1994;134:924–34.


The Angelina Jolie Effect in Jewish Law: Prophylactic Mastectomy and Oophorectomy in BRCA Carriers
Sharon Galper Grossman
Rambam Maimonides Med J 2015 October;6(4):e0037   http://www.rmmj.org.il/(S(zgs5agxe5czk4nak0stlsn1c))/Pages/Article.aspx

Background: Following the announcement of actress Angelina Jolie’s prophylactic bilateral mastectomies and subsequent prophylactic oophorectomy, there has been a dramatic increase in interest in BRCA testing and prophylactic surgery.

Objective: To review current medical literature on the benefits of prophylactic mastectomy and oophorectomy among BRCA-positive women and its permissibility under Jewish law.
Results: Recent literature suggests that in BRCA-positive women who undergo prophylactic oophorectomy the risk of dying of breast cancer is reduced by 90%, the risk of dying of ovarian cancer is reduced by 95%, and the risk of dying of any cause is reduced by 77%. The risk of breast cancer is further reduced by prophylactic mastectomy. Prophylactic oophorectomy and prophylactic mastectomy pose several challenges within Jewish law that call into question the permissibility of surgery, including mutilation of a healthy organ, termination of fertility, self-wounding, and castration. A growing number of Jewish legal scholars have found grounds to permit prophylactic surgery among BRCA carriers, with some even obligating prophylactic mastectomy and oophorectomy.
Conclusion: Current data suggest a significant reduction in mortality from prophylactic mastectomy and oophorectomy in BRCA carriers. While mutilation of healthy organs is intrinsically forbidden in Jewish law, the ability to preserve human life may contravene and even mandate prophylactic surgery.
INTRODUCTION In May 2013, the widely acclaimed Hollywood celebrity, Angelina Jolie, published an op-ed in The New York Times announcing that her mother, grandmother, and aunt had had cancer, that she had tested positive for the BRCA mutation, and that she had undergone bilateral prophylactic mastectomies to prevent breast cancer.1 This announcement led to what oncologists refer to as “The Angelina Jolie Effect,” a more than doubling in the demand for BRCA testing in women who would not otherwise have gone for testing, but were at high risk for carrying the mutation based on family history and therefore should have undergone genetic testing.2 In March 2015, Angelina Jolie published a second oped in The New York Times disclosing that she had undergone laparoscopic oophorectomy, removal of the ovaries to prevent ovarian cancer, and that she was receiving hormone replacement therapy to prevent the side-effects of premature menopause.3

SCIENTIFIC BACKGROUND Hereditary breast cancer accounts for 5%–10% of all breast cancer.4–7 The vast majority of inherited breast cancers are due to mutations in two breast cancer genes referred to as BRCA1 and 2.6 The risks of developing breast and ovarian cancer are higher in carriers of the BRCA1 mutation compared to carriers of BRCA2.8 In addition, cancer is more likely to occur at a younger age in carriers of BRCA1 mutations than in carriers of BRCA2. An average woman has a 12% lifetime risk of developing breast cancer.9 In a recent population-based study of Ashkenazi Jews in Israel, regardless of family history, the risks of developing breast cancer among BRCA1 and 2 carriers were 60% and 40%, respectively, and the risks of developing ovarian cancer were 53% and 62%, respectively.10 These results are consistent with the findings of a meta-analysis of 10 studies of patients in high-risk clinics which reported that the risks of developing breast cancer by the age of 70 in BRCA1 and 2 carriers are 57% and 49%, respectively, and the risks of ovarian cancer are 40% and 18%, respectively.8 These risks are significantly higher among women born more recently than among women born earlier, a birth cohort effect presumably due to modifications in non-genetic factors such as earlier menarche and later childbearing.10
Possible interventions for BRCA carriers might include increased surveillance, chemoprevention, and prophylactic surgery. Surveillance for breast cancer has consisted of MRI and mammogram beginning at age 25 or individualized to 10 years before the first cancer diagnosed in the family. While the addition of MRI to mammogram increases cancer detection rates and diagnoses cancer at an earlier stage, this strategy has not been shown to prolong survival in BRCA carriers.11–14 Surveillance for ovarian cancer has consisted of trans-vaginal ultrasound and blood tests for elevated tumor markers such as CA-125. However, this strategy has not been found to be effective.15,16 In fact, the National Cancer Institute does not recommend surveillance for ovarian cancer among BRCA carriers.
Another approach to reducing the risk of cancer among BRCA carriers is chemoprevention: tamoxifen to reduce the risk of breast cancer, and oral contraceptive pills to reduce the risks of ovarian cancer. Tamoxifen appears to be effective in reducing breast cancer in carriers of BRCA2 but not in carriers of BRCA1.17,18 The differential effect of tamoxifen may be due to the differential expression of the estrogen receptor in tumors of BRCA carriers. The BRCA1 carriers tend to develop estrogen receptor-negative breast cancers which do not depend on estrogen to grow, and the BRCA2 carriers tend to develop breast cancers that are estrogen receptor-positive and depend on estrogen to grow.19–21 Chemoprevention with tamoxifen has not been shown to prolong survival in BRCA2 carriers.17,18 Oral contraceptive pills have been shown to reduce the chances of developing ovarian cancer in BRCA carriers by 50% without increasing the risk of breast cancer, but this intervention has not been shown to reduce the chances of dying of ovarian cancer.22
PROPHYLACTIC SURGERY Prophylactic surgery consists of mastectomy, removal of the breasts to prevent breast cancer, and oophorectomy, removal of the ovaries to prevent ovarian cancer. Prophylactic mastectomy reduces the chances of developing breast cancer by 90% or more.23–30 In one series, there were no cases of breast cancer 3 years after prophylactic surgery.29 Prophylactic oophorectomy reduces the chances of developing ovarian cancer by 80% and can also reduce the chances of developing breast cancer by 50%.31–35 Carriers of the BRCA mutation may opt for mastectomy alone with surveillance of the ovaries (as Angelina Jolie chose to do between 2013 and 2015), prophylactic oophorectomy alone which will reduce the chances of developing both breast and ovarian cancer, or prophylactic mastectomy and oophorectomy (which Angelina Jolie ultimately chose to do). The ideal age to perform prophylactic oophorectomy is not known, but the recommendation is to complete childbearing by age 35–40 and then undergo prophylactic surgery as there is concern that delaying oophorectomy would increase the chances of developing ovarian cancer.35 In addition, the magnitude of the protective effects of oophorectomy in reducing the chances of breast cancer is greater the younger the age of oophorectomy.29,36 Oophorectomy at this age does cause premature menopause; however, as Angelina Jolie has illustrated, these symptoms can safely be managed with short-term hormone replacement therapy.37–39
How do BRCA carriers cope with prophylactic surgery? Emerging data would suggest that overall quality of life for BRCA carriers who undergo prophylactic surgery is not compromised by surgery. The BRCA carriers who undergo prophylactic surgery report high satisfaction with surgery and less cancer worry than BRCA carriers who opt for surveillance.40,41 However, prophylactic surgery may be associated with sexual dysfunction and menopausal symptoms which can be addressed by proper medical intervention. Although it was initially thought, based on theoretical modeling, that BRCA carriers who underwent prophylactic surgery would live longer than those who opted for surveillance, this was not based on actual prospective patient data.42 Recently, a number of studies have confirmed that BRCA carriers who undergo prophylactic surgery live longer than those who undergo surveillance.29,35,43 In one series, the chances of dying of ovarian cancer were reduced by 95% among BRCA carriers who opted for prophylactic oophorectomy compared to carriers who opted for surveillance, the chances of dying of breast cancer were reduced by 90%, and the chances of dying of any cause were reduced by 70%.29 Previously, it was assumed that prophylactic oophorectomy would reduce the chances of a BRCA carrier dying of ovarian cancer but that inducing premature menopause would be harmful and negate any beneficial effects of preventing ovarian cancer. Physicians believed that the BRCA carrier who opted for prophylactic oophorectomy was “trading” ovarian cancer for a host of new medical problems associated with entering premature menopause for no “net” benefit. Yet, the most recent studies show that, overall, BRCA carriers who undergo prophylactic surgery live longer than those who opt for surveillance, demonstrating that, regardless of what medical problems premature menopause may cause, the net effect of prophylactic surgery is that carriers who undergo such surgery live longer. The medical benefits clearly outweigh the risks.
HALACHIC ISSUES From a Halachic perspective, there are several concerns arguing against prophylactic surgery (Table 1). First of all, prophylactic surgery involves mutilation of a healthy organ. Secondly, removing a healthy organ—especially one that defines a woman’s sexuality and her appearance—may cause significant psychological distress, although the available quality of life data would suggest that from a psychological perspective this surgery is well tolerated.40,41
In addition, there are two other potential Halachic concerns regarding removal of ovaries. First, removal of ovaries may prevent the BRCA carrier from fulfilling the mitzvah to “be fruitful and multiply” (pru u’revu). Second, removal of ovaries may violate the prohibition against castration (sirus). Arguing in favor of prophylactic surgery are new, emerging, compelling medical data showing that BRCA carriers who undergo prophylactic surgery are less likely to die of cancer and more likely to live longer than women who opt for surveillance. Angelina Jolie’s decision prophylactically to remove her healthy breasts and most recently her healthy ovaries raises several Halachic questions including the following: (1) is there a Halachic obligation to prevent disease; (2) is it permitted for a BRCA carrier to remove a healthy organ; (3) does prophylactic surgery violate the prohibition against harming one’s body (chovel); (4) does prophylactic oophorectomy prevent the BRCA carrier from fulfilling the mitzvah to procreate; (5) does prophylactic oophorectomy violate the prohibition against castration; (6) is it permitted to perform prophylactic surgery in a BRCA carrier; (7) is a BRCA carrier obligated by Halacha to undergo prophylactic surgery; and, lastly, (8) are we as a Jewish society, particularly in Israel, obligated to pay for prophylactic surgery in BRCA carriers?
In summary, there is a clear Halachic obligation to prevent disease. It is permitted to remove a healthy body part to prevent disease in the future. Prophylactic oophorectomy interferes with obligations to procreate and prohibitions of castration. Oophorectomy after completing childbearing helps eliminate issues relating to “procreation.” When performed after menopause, prophylactic oophorectomy may obviate the prohibition against castration. Ultimately, saving a life overrides castration and any prohibitions including the prohibition against harming one’s body. Given the emerging data favoring prophylactic surgery, a growing number of Jewish arbiters believe that prophylactic surgery is Halachically permitted, with some positing that a BRCA carrier is obligated to undergo prophylactic surgery.
Angelina Jolie’s Status in Judaism Angelina Jolie’s very public medical journey has increased awareness of the BRCA mutation and the demand for testing in high-risk women who would not otherwise have been tested. In addition, she has increased interest in potentially life-saving prophylactic surgery. It is not possible to measure how many lives she has saved by making her very personal, medical odyssey public. Regardless of her other behaviors and politics, her decision to publicize her status as a BRCA carrier and her decisions to undergo prophylactic surgery make her worthy of the description in Sanhedrin, “Whoever saves one Jewish life is considered to have created an entire world.”63
Angelina Jolie has created many worlds, and for this we as a Jewish people must be eternally grateful.

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Mutation D538G – a novel mechanism conferring acquired Endocrine Resistance causes a change in the Estrogen Receptor and Treatment of Breast Cancer with Tamoxifen

Reporter: Aviva Lev-Ari, PhD, RN


Mutation Linked to Resistance in Breast Cancer

Published: Nov 12, 2013

By Charles Bankhead, Staff Writer, MedPage Today
Reviewed by F. Perry Wilson, MD, MSCE; Instructor of Medicine, Perelman School of Medicine at the University of Pennsylvania and Dorothy Caputo, MA, BSN, RN, Nurse Planner


A mutation on the estrogen receptor altered tamoxifen bindings and conferred tumor cell resistance to the breast cancer agent, suggesting a pathway to treatment resistance, investigators reported.

The D538G mutation appeared in liver metastases but not primary breast tumors of women treated with hormonal therapy, according to Ido Wolf, MD, of the University of Tel Aviv in Israel, and colleagues.

Laboratory studies suggested that D538Gcauses a change in the estrogen receptor that “disrupts the interaction between the receptor and either estrogen or tamoxifen, bur mimics the conformation of the activated receptor,” they wrote online inCancer Research. “Studies in cell lines confirmed ligand-independent, constitutive activity of the mutated receptor.”

“Taken together, these data indicate the mutation D538G as a novel mechanism conferring acquired endocrine resistance.”

About three-fourths of all breast cancers express estrogen receptor-alpha. Targeting the receptor with tamoxifen, or another hormonal therapy, disrupts signaling that reduces levels of the functional protein produced that binds to the receptor or inhibits the receptor.

Some patients with metastatic breast cancer do not respond to any form of endocrine treatment (primary resistance). Almost all patients who initially respond to endocrine therapy eventually develop resistance to the therapy (acquired resistance).

Several mechanisms of acquired resistance have been identified, including reduced expression of estrogen receptor alpha, altered activity of regulatory proteins, and increased activity of growth factor signaling pathways that help drive tumor growth and progression, the authors noted.

Protein mutation occurs with some regularity in tumorigenesis, but fewer than 1% of primary breast tumors develop mutations in estrogen receptor-alpha. No previous studies had shown that acquired mutations in estrogen receptor-alpha might play a role in the development of resistance to hormonal therapy.

As part of their search for cancer-related genes, Wolf and colleagues examined tumor specimens from 13 patients with metastatic breast cancer that had proven unresponsive to multiple lines of therapy. Genetic analysis showed that metastases from five patients had developed mutations in estrogen receptor-alpha, resulting in the substitution of aspartic acid to glycine at position 538 of the receptor gene.

“Importantly, the mutation was not detected in the primary tumors obtained prior to endocrine treatment,” the authors noted in their discussion of the findings.

Two previous activating mutations of estrogen receptor-alpha have been identified, but neither has been linked to resistance to hormonal therapy for breast cancer. Moreover, no acquired mutations (not present in the primary tumor) had been identified previously.

A limitation of this study was that the women were “highly selected, heavily pretreated patients and may not represent the general population of patients with breast cancer,” the authors pointed out.

“The actual prevalence of the D538G mutation needs to be determined in large cohorts of patients,” Wolf and colleagues concluded. “If indeed the mutation is identified in a significant proportion of patients, direct testing of it may be an easy and cheap method to predict response to hormonal therapy.”

The study was supported by the Israel Cancer Association, Margaret Stultz Foundation, Sackler Faculty of Medicine, and the Israel Science Foundation.

Wolf reported no conflicts of interest. One or more co-authors reported relationships with Oncotest-Teva Pharmaceuticals, Foundation Medicine, and Novartis.




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Close relative of a breast cancer survivor had more than a two-fold increased risk of ovarian cancer compared to women with no family history of breast cancer.

Reporter: Aviva Lev-Ari, PhD, RN

July 24, 2013

Family Cancer Ties Run Deep

Author Info

Reviewed by:

Joseph V. Madia, MD


Risks of different cancer types higher in first degree relatives of cancer survivors

(dailyRx News) If a woman’s mother, sister or daughter has had breast cancer, she has increased risks of developing the disease herself. But is this woman at greater risk of other types of cancer, too?

A recent study has found that a family history of cancer may increase the risk of close relatives developing the same type of cancer as well as different forms of the disease.

For example, this study showed that the close relative of a breast cancer survivor had more than a two-fold increased risk of ovarian cancer compared to women with no family history of breast cancer.

“Alert your healthcare providers about any family history of cancer.”

Eva Negri, MatSciD, head of the Laboratory of Epidemiologic Methods at the Mario Negri Institute for Pharmacological Research in Milan, Italy, was the corresponding author of the study.

The goal of this study was to look at the cancer histories of close relatives, particularly first-degree relatives (siblings, parents and children), of cancer survivors.

Dr. Negri and colleagues from Switzerland and France analyzed case-control studies (cancer cases versus healthy comparisons) on thirteen different types of cancer conducted between 1991 and 2009.

Cancer types included mouth and throat, nasal, larynx (voicebox), esophageal (tube between throat and stomach), stomach, colorectal, liver, pancreas, throat, breast, endometrial, ovary, prostate and kidney.

Data was collected on more than 12,000 cancer cases and more than 11,000 comparisons.

Along with information on family history of cancer, the researchers looked at age of diagnosis, body shape, lifestyle habits such as diet, smoking and alcohol use and personal history including reproductive history, use of birth control and hormonal therapies.

After accounting for all other factors, the researchers found the following:

  • Women with a family history of colorectal cancer had a 1.5-fold increased risk of developing breast cancer.
  • Female first-degree relatives of a breast cancer survivor had a 2.3-fold increased risk of ovarian cancer.
  • Male first-degree relatives of a bladder cancer survivor had a 3.4-fold increased risk of prostate cancer.
  • Close relatives of someone who had mouth (oral) or throat cancer had a four-fold increased risk of esophageal cancer.
  • People with a first-degree relative who had cancer of the larynx had a 3.3-fold increased risk of developing oral or throat cancer.
  • If cancer was diagnosed in an individual before the age of 60, the risk of close family members developing a different type of cancer was greater.

“Our results point to several potential cancer syndromes that appear among close relatives and that indicate the presence of genetic factors influencing multiple cancer sites,” the authors wrote.

Dr. Negri said in a prepared statement, “These findings may help researchers and clinicians to focus on the identification of additional genetic causes of selected cancers and on optimizing screening and diagnosis, particularly in people with a family history of cancer at a young age.”

This study was published July 24 in the Annals of Oncology.

This work was supported by the Italian Association for Cancer Research, the Italian Ministry of Education and the Swiss League Against Cancer.

No conflicts of interest were disclosed.



Reviewed by:
Review Date:

July 25, 2013

Last Updated:

July 25, 2013



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Reporter: Aviva Lev-Ari, PhD, RN



Changes in breast density point to tamoxifen‘s effectiveness

By Kate Madden Yee, AuntMinnie.com staff writer

April 22, 2013 — If women being treated with tamoxifen for breast cancer see their breast density drop, they may have a 50% lower risk of dying from the disease, according to a new study by Swedish researchers published online April 22 in the Journal of Clinical Oncology.

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In a study that hints at a role for breast density measurement in predicting therapy response, researchers at Karolinska Institutet in Stockholm found that women who had a relative reduction of more than 20% in the absolute dense area of their breast tissue during the course of tamoxifen treatment had a 50% reduction in breast cancer mortality over a span of 15 years, compared with women who had little or no change (JCO, April 22, 2013).

Tamoxifen is usually given over five years to prevent the recurrence of breast cancer in women who have completed their primary treatment. The researchers conducted the study to assess whether there was a link between reduced tissue density and the effectiveness of tamoxifen therapy.

Dr. Per Hall

Dr. Per Hall from Karolinska Institutet.

No method has been available for assessing which women are likely to respond to tamoxifen and not relapse with breast cancer, according to Dr. Per Hall and colleagues.

“To the best of our knowledge, this is the first time mammographic density change has been used as a prognostic marker of response to tamoxifen,” Hall and colleagues wrote. “We observed that women treated with tamoxifen who experienced mammographic density reduction were associated with substantially better long-term breast-cancer-specific survival. If validated, mammographic density change has the potential to be an early marker for therapy response.”

For the study, Hall and colleagues included data collected in Sweden between 1993 and 1995 from 974 postmenopausal patients with breast cancer who had both baseline and follow-up mammograms. Of these, 474 patients received tamoxifen treatment and 500 did not. The team measured mammographic density using a statistical method that expressed the data as absolute dense area.

During the follow-up period, 121 patients (12.4%) died from breast cancer. But women who were treated with tamoxifen and who experienced a relative density reduction of more than 20% had a 50% lower risk of death, compared with women whose breast density didn’t change, the team found.

In the group that was not treated with tamoxifen, there was no statistically significant association between mammographic density change and survival, and the survival advantage was not found when absolute dense areas at baseline or follow-up were evaluated separately.

The findings come on the heels of a recommendation issued on April 15 from the U.S. Preventive Services Task Force (USPSTF) regarding the use of tamoxifen and a related drug, raloxifene, as preventive measures against breast cancer in asymptomatic women. The USPSTF suggests that women who are at increased risk for breast cancer talk to their physician about the potential benefits and harms of the drugs to prevent breast cancer; women who are not at increased risk should not use them.

If further research confirms the Swedish group’s findings, mammographic density change has the potential to be an early marker for therapy response, Hall and colleagues wrote. In fact, given ongoing developments in automatic algorithms for mammographic density measurement, using density change to monitor the effectiveness of treatment could be a cost-effective clinical tool.

“What’s needed is accurate measurement of mammographic density,” Hall said in a statement released by the Karolinska Institutet. “Measuring changes in density can be a simple and cheap means of assessing the effect of the treatment. If a patient is not responding to tamoxifen, maybe they should be given a different drug.”

Related Reading

ARRS: Breast US spots missed cancers in dense breasts, April 18, 2013

AHRA backs Are You Dense Advocacy, April 10, 2013

Calif. breast density bill goes into effect, April 1, 2013

Rads judge breast density the same for digital, analog mammo, February 28, 2013

Yearly screening breast US benefits women with dense tissue, December 4, 2012
Copyright © 2013 AuntMinnie.com



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Cancer Genomic Precision Therapy: Digitized Tumor’s Genome (WGSA) Compared with Genome-native Germ Line: Flash-frozen specimen and Formalin-fixed paraffin-embedded Specimen Needed

Curator: Aviva Lev-Ari, PhD, RN

Dr. Charles Swanton, Cancer Research, UK’s London Research Institute explained in his March 29, 2013 interview for Science, that the cancer treatments often fail as a result of the increasing evidence that tumors contain a heterogeneous mix of cells – tissue tagged with colored fluorescent markers for specific molecular changes sh0ws that not all cells in a tumor are the same.

http://www.sciencemag.org SCIENCE vol 339, 3/29/2013, 1543-1545

His team sequenced DNA taken from different parts of a patient’s kidney tumor, the sequence of each part was different. Severaal genetic changes were shared throughout the original tumor mass and iother tumors, or metastases, most were present in only some parts, suggesting that tumors host diverse populations of cells. some of these cells may be resistant to a treatment and continue to grow.

The findings presented below from Swanton’s Lab and from Polyak’s Lab demonstrate that intra-tumor heterogeneity as revealed by genome sequencing applied as Multiregion Sequencing might suggest that the current best practice in Oncology, of a single biopsy, must be abandoned for the sake of a new and more promising practice of multiple biopsies of the same tumor.

Kornelia Polyak’s Lab at Dana-Farber Cancer Institute, dedicated to the molecular analysis of human breast cancer


Our goals are to:

  • Better understand the molecular evolution of human breast tumors
  • Use this knowledge to improve the clinical management of breast cancer patients

Project 1: Breast tumor evolution

Modeling clonal evolution in mouse xenograft models

Cancers develop as a result of somatic evolution. Deciphering the evolutionary dynamics behind this should provide a more accurate understanding of how cancers arise and enable more intelligent approaches toward anti-cancer therapies. However, this area receives almost no experimental attention, and our understanding of clonal evolution in cancers is very rudimentary. To address this deficiency, we have developed a mouse xenograft model of human breast cancers that allows us to follow dynamics of clonal competition in genetically heterogeneous tumors.

Intratumor heterogeneity and metastasis

Metastatic dissemination of cancer cells is the most prominent cause of death due to breast cancer. Recent work in this field has established that the progression of metastatic invasion from the primary tumor to distant locations (such as bone, lungs, and brain) depends on heterogeneous interactions of cancer cells with each other and with cells composing the microenvironment. We aim to elucidate some of the factors and mechanisms that influence metastatic co-operation between cancer cells and their environment in order to fully understand the metastatic cascade and aid in the development of therapies that address this phenomenon.

Diversity in human breast tumors

Intra-tumor genetic and phenotypic diversity may predict the risk of breast cancer progression and response to treatment. To deepen our understanding of these factors, we have been defining intra-tumor diversity using immuno-FISH and ecological models in breast tumors at different progression stages (i.e., in situ, invasive, metastatic), and before and after chemotherapy or targeted (e.g., antu-Her2) treatment.

Project 2: The role of the tumor microenvironment in breast cancer

Interrogating consequences of interactions between breast carcinoma cells and tumor fibroblasts

While it is becoming increasingly apparent that interactions between carcinoma cells and tumor stroma are an essential part of tumor biology, our understanding of this crosstalk is far from complete. Using organotypic 3D culture models, we are interrogating mutual changes in transcriptome, metabolome, and phospho-proteome that result from the interaction between breast carcinoma cells and primary breast tumor-associated fibroblasts.

Myoepithelial cells and leukocytes in DCIS

The progression from in situ to invasive carcinoma is a key but poorly understood step of breast tumorigenesis, characterized by loss of the myepithelial cell layer and basement membrane. We hypothesize that the differentiation of bipotential mammary epithelial progenitors to myoepithelial cells is progressively inhibited by signals coming from tumor epithelial cells and stromal cells, such as leukocytes, leading to their eventual disappearance. Project objectives include:

  • Defining normal myoepithelial cell differentiation and its abnormalities in DCIS
  • Characterizing the role of immune cells in myoepithelial cell differentiation during breast carcinoma progression using in vivo and in vitro model systems and human breast tissue

The completion of this project will increase our understanding of the role of myoepithelial and immune cells in breast cancer, and may also provide new targets for breast cancer treatment via abnormally expressed paracrine signaling in the tumor microenvironment.

Project 3: Epigenetics in breast cancer risk and tumor development

Pregnancy study

Human epidemiological and experimental data in rodent models suggest that full-term pregnancy in early adulthood decreases the risk of estrogen receptor positive (ER+) breast cancer in post-menopausal women; however, the underlying mechanism is largely unknown. We hypothesized that the cancer-preventive effects of parity may be due to alterations in the number or properties of mammary epithelial progenitor/stem cells that are thought to be the cell-of-origin of breast cancer, rendering them less susceptible to oncogenesis. To test this hypothesis, we analyzed the relative frequency and comprehensive molecular profiles of four distinct cell types (CD24+ luminal, CD10+ myoepithelial, lin-/CD24-/CD44+ progenitor-enriched, and stromal fibroblasts) isolated from normal breast tissue of premenopausal nulliparous and parous women. Based on the comprehensive analysis of gene expression, DNA, and histone H3 K27 trimethylation profiles of these cell types, we determined that the most significant changes occurred in lin-/CD24-/CD44+ progenitor-enriched cells. The activity of many genes and pathways involved in development, differentiation, and cell cycle regulation are decreased in parous women that may contribute to their decreased breast cancer risk. We also identified a parity-associated gene signature that predicted clinical outcome in breast cancer patients diagnosed with ER+ tumors.

The role of DNA methylation in mouse mammary gland development

The mouse mammary gland is a useful model system for understanding factors that regulate mammary development. We are pursuing molecular characterization of the different cell types that comprise the mammary epithelium of the mouse. Based on the varying proportional distributions we observe in the mature, progenitor, and stem cell populations of the mammary gland during different life stages, we seek to understand the underlying molecular cues that maintain cell type identities and direct cellular distribution changes by studying the gene expression and epigenetic properties of distinct cell populations during puberty and pregnancy, stages during which there is dramatic tissue remodeling in the mammary gland. Furthermore, with the use of in vitro and in vivo mouse models for the functional characterization of maintenance DNA methylation, we are characterizing potential active roles of this important epigenetic mark in directing cell fate in the mammary gland.

Histone modifying enzymes as new therapeutic targets

The differentiation of normal stem cells and the development of normal tissue are controlled by epigenetic mechanisms. Abnormalities in these processes play a role in the initiation and progression of tumors and intra-tumor diversification of cancer cells. A number of histone-modifying genes were found to be mutated in breast and other cancers, implying that these genes may represent novel therapeutic targets and biomarkers. We have recently reported the characterization of cell-type specific patterning of histone and DNA methylation in normal breast tissues. We developed modified chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-Seq) protocol which enables us to investigate the epigenetic status genome-wide, using limited numbers of cells purified from human breast tissue samples. Currently, we are using various genomic profiling and functional studies to validate several histone demethylases as potential therapeutic targets in breast cancer.

Determinants of basal-like and luminal breast cancer cell phenotypes

Basal-like and luminal breast tumors have distinct molecular profiles and clinical behavior, yet the mechanisms underlying these differences are poorly defined. We investigated the potential role of genetic factors in determining these distinct phenotypes and their inheritance pattern by generating somatic cell fusions between basal-like and luminal breast cancer cells and analyzing their molecular profiles and functional characteristics. Based on the molecular profiles, we identified candidate key transcriptional and epigenetic determinants of basal-like and luminal cell phenotypes. We are further characterizing these genes using functional genomics approaches.

Project 4: Emerging therapeutic targets in breast cancer

Amplified kinases and novel targets in breast cancer

Kinase inhibitors have been one of the most successful drugs for cancer treatments, but their efficacies in patients are still not satisfactory. We have identified novel kinases amplified in breast cancer, and are using functional genomic approaches to validate them as therapeutic targets.

Novel therapeutic targets in triple negative breast cancer

We have conducted an shRNA cell viability screen of 1,576 candidate genes differentially expressed between CD44+CD24- stem cell-like and CD44-CD24+ more differentiated luminal breast cancer cells. These shRNA were further tested across 14 breast cancer cell lines, thereby generating a list of 15 genes of high interest as candidate therapeutic targets against CD44+CD24- cells, including IL6, CXCL3, PTGIS, IGFBP7, PFKFB3 and HAS1. We have followed up and validated the Il6/Jak2/Stat3 signaling pathway in further detail and demonstrated that JAK2 inhibitors may effectively inhibit the growth of breast tumors that have activation of this pathway as determined based on expression of phospho-Stat3 (pStat3). Based on our preclinical data, a clinical trial testing the efficacy of Jak2 inhibitors in pStat3+ breast tumors (enriched in BLBC) is being initiated at DFCI. More recently we also found that a high fraction of inflammatory breast cancer (IBC) are also positive for pStat3, and thus, may respond to JAK kinase inhibition. Besides the JAK/Stat3 pathway, other potentially promising targets include CXCR2, PTGIS, and HAS1. We are conducting preclinical studies validating these genes and their combination as potential new therapeutic strategies in breast cancer.


Swanton’s results was published in NEJM on March 8, 2012. 143 citations followed by year end.


Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

Marco Gerlinger, M.D., Andrew J. Rowan, B.Sc., Stuart Horswell, M.Math., James Larkin, M.D., Ph.D.,

David Endesfelder, Dip.Math., Eva Gronroos, Ph.D., Pierre Martinez, Ph.D., Nicholas Matthews, B.Sc.,

Aengus Stewart, M.Sc., Patrick Tarpey, Ph.D., Ignacio Varela, Ph.D., Benjamin Phillimore, B.Sc., Sharmin Begum, M.Sc.,

Neil Q. McDonald, Ph.D., Adam Butler, B.Sc., David Jones, M.Sc., Keiran Raine, M.Sc., Calli Latimer, B.Sc.,

Claudio R. Santos, Ph.D., Mahrokh Nohadani, H.N.C., Aron C. Eklund, Ph.D., Bradley Spencer-Dene, Ph.D.,

Graham Clark, B.Sc., Lisa Pickering, M.D., Ph.D., Gordon Stamp, M.D., Martin Gore, M.D., Ph.D., Zoltan Szallasi, M.D.,

Julian Downward, Ph.D., P. Andrew Futreal, Ph.D., and Charles Swanton, M.D., Ph.D.

Abstr act

From the Cancer Research UK London

Research Institute (M. Gerlinger, A.J.R.,

S.H., D.E., E.G., P.M., N.M., A.S., B.P.,

S.B., N.Q.M., C.R.S., B.S.-D., G.C., G.S.,

J.D., C.S.), Royal Marsden Hospital Department

of Medicine ( J.L., M.N., L.P.,

G.S., M. Gore), Wellcome Trust Sanger

Institute (P.T., I.V., A.B., D.J., K.R., C.L.,

P.A.F.), Barts Cancer Institute at the

Barts and the London School of Medicine

and Dentistry (M. Gerlinger), and the University

College London Cancer Institute

(C.S.) — all in London; the Technical University

of Denmark, Lyngby (A.C.E., Z.S.);

and Harvard Medical School, Boston (Z.S.).

Address reprint requests to Dr. Swanton at

the Cancer Research UK London Research

Institute, Translational Cancer Therapeutics

Laboratory, 44 Lincoln’s Inn Fields,

London WC2A 3LY, United Kingdom, or

at charles.swanton@cancer.org.uk.

Drs. Gerlinger, Larkin, Gronroos, Martinez,

and Swanton and Mr. Rowan, Mr. Horswell,

Mr. Endesfelder, Mr. Matthews, and

Mr. Stewart contributed equally to this


N Engl J Med 2012;366:883-92.

Copyright © 2012 Massachusetts Medical Society.


Intratumor heterogeneity may foster tumor evolution and adaptation and hinder

personalized-medicine strategies that depend on results from single tumor-biopsy



To examine intratumor heterogeneity, we performed exome sequencing, chromosome

aberration analysis, and ploidy profiling on multiple spatially separated samples obtained

from primary renal carcinomas and associated metastatic sites. We characterized

the consequences of intratumor heterogeneity using immunohistochemical analysis,

mutation functional analysis, and profiling of messenger RNA expression.


Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to

69% of all somatic mutations not detectable across every tumor region. Intratumor

heterogeneity was observed for a mutation within an autoinhibitory domain of the

mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP

phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro.

Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes

converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct

and spatially separated inactivating mutations within a single tumor, suggesting

convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis

were detected in different regions of the same tumor. Allelic composition and

ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor

samples from four tumors harboring divergent allelic-imbalance profiles and with

ploidy heterogeneity in two of four tumors.


Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape

portrayed from single tumor-biopsy samples and may present major challenges to

personalized-medicine and biomarker development. Intratumor heterogeneity, associated

with heterogeneous protein function, may foster tumor adaptation and therapeutic

failure through Darwinian selection. (Funded by the Medical Research Council

and others.)

n engl j med 366;10 nejm.org march 8, 2012

Sci Transl Med 28 March 2012:
Vol. 4, Issue 127, p. 127ps10
Sci. Transl. Med. DOI: 10.1126/scitranslmed.3003854


Intratumor Heterogeneity: Seeing the Wood for the Trees

  1. Timothy A. Yap1*,
  2. Marco Gerlinger2,3*,
  3. P. Andrew Futreal4,
  4. Lajos Pusztai5 and
  5. Charles Swanton2,6†

+Author Affiliations

  1. 1Department of Medicine, Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey SM2 5PT, UK.

  2. 2Translational Cancer Therapeutics Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK.

  3. 3Barts Cancer Institute, Barts and The London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK.

  4. 4Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

  5. 5Department of Breast Medical Oncology, Division of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

  6. 6University College London Cancer Institute, Huntley Street, London WC1E 6BT, UK.

  7. *These authors contributed equally to this work.
  1. Corresponding author. E-mail: charles.swanton@cancer.org.uk


Most advanced solid tumors remain incurable, with resistance to chemotherapeutics and targeted therapies a common cause of poor clinical outcome. Intratumor heterogeneity may contribute to this failure by initiating phenotypic diversity enabling drug resistance to emerge and by introducing tumor sampling bias. Envisaging tumor growth as a Darwinian tree with the trunk representing ubiquitous mutations and the branches representing heterogeneous mutations may help in drug discovery and the development of predictive biomarkers of drug response.

Citation: T. A. Yap, M. Gerlinger, P. A. Futreal, L. Pusztai, C. Swanton, Intratumor Heterogeneity: Seeing the Wood for the Trees. Sci. Transl. Med. 4, 127ps10 (2012).


In Science Translational Medicine

  • EDITORIAL:CANCERWinning the War: Science Parkour

    • Bert Vogelstein and
    • Kenneth W. Kinzler

    Sci Transl Med 28 March 2012 4:127ed2


  • Development of Therapeutic Combinations Targeting Major Cancer Signaling PathwaysJCO 20 April 2013 31:1592-1605
  • A tale of two approaches: complementary mechanisms of cytotoxic and targeted therapy resistance may inform next-generation cancer treatmentsCarcinogenesis 1 April 2013 34:725-738
  • Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamicsProc. Natl. Acad. Sci. USA 5 March 2013 110:4009-4014
  • Accelerating Cancer Therapy Development: The Importance of Combination Strategies and Collaboration. Summary of an Institute of Medicine WorkshopClin. Cancer Res. 15 November 2012 18:6101-6109
  • Pluripotent Stem Cell-Based Cancer Therapy: Promise and ChallengesSci Transl Med 28 March 2012 4:127ps9


Topol on the Cancer Clinic of the Future

Hello. I’m Dr. Eric Topol, director of the Scripps Translational Science Institute and Editor-in-Chief of Medscape. In this series, The Creative Destruction of Medicine, named for the book I wrote, I am trying to zoom in on critical aspects of how the digital world can create better healthcare.

Cancer care is rapidly changing, if we think about where it was some years ago as it was really beautifully archived in a book by Sid Mukherjee, MD, The Emperor of All Maladies,[1] and to where we can go in the future. Just launched recently, for example, was MD Anderson Cancer Center’s Moon Shots program in cancer care.[2] The Moon Shots program is perhaps, because of genomics, digitizing the genome of the tumor, comparing it with the genome-native germ line. This gives us an opportunity we never had before.

So what is the cancer clinic of the future going to look like, because it’s just starting to get developed today? For example, when we have an individual presenting for a new diagnosis of cancer, we have to move away from fine-needle aspiration and minimal tissue; we need real tissue to be able to process it properly. Not only do we need the formalin-fixed paraffin-embedded (FFPE) specimen, but we also need another type of FF — that is, flash-frozen specimens so that we can then whole-genome sequence this tissue.

Now, when that is done at the primary diagnosis and done within hours and analyzed with the appropriate software algorithms, we could get the driver mutations nailed within 24 hours from the diagnosis. This can set up remarkably precise therapy that can be given to the patient on the basis of that individual’s tumor. There are no 2 different cancers that are the same anywhere. Just like there are no 2 individuals who have the same DNA, that’s the same for a tumor.

One of the issues that we have to confront is that there’s a lot of intratumor heterogeneity. We need multiple samples to sequence from the tumor, and if there’s already a metastatic lesion, we need a sample of that as well. Multiple sequencing, frozen tissue, genome-driven guided therapy — right from the get-go — is what we need. That’s not what we have today, but that’s where we can go in the future of cancer genomic medicine. It’s really an exciting opportunity. It has to be validated.

The cancer drugs that are used today are remarkably expensive, and what’s fascinating is to see — and this is a recurrent theme — is that a drug being used, for example, for renal carcinoma can also be used for leukemia. There was a classic 3-part article on the front page of the New York Times [3] that exemplified some of the stories along those lines.

It’s a story about mutations — a war on mutations, not a war on cancer — and this type of cancer clinic in the future can take us there but there’s going to have to be a whole different look with respect to the way that we take samples of the tumor. We need much more tissue, and to use frozen tissue so that we don’t have to bootstrap the FFPE (that paraffin-embedded specimen) and only get a couple of hundred genes or coding elements, but in fact get a whole genome from the flash-frozen specimen. That’s really important, and we have to move in that direction — get more tissue in order to account for the heterogeneity that we know exists. And we have to do deep sequencing of that frozen tissue in order to get the driver mutations identified, and also be able to anticipate where relapses can occur downstream.

That is precision therapy. This exemplifies the future of cancer genomic medicine, and it will be really interesting to see how that plays out in these cancer clinics of the future.

Thanks so much for joining us for this segment, and stay tuned for more from The Creative Destruction of Medicine series.


  1. Mukherjee S. The Emperor of All Maladies: A Biography of Cancer. New York: Scribner; 2010. The 2011 Pulitzer Prize Winners: General Nonfiction. http://www.pulitzer.org/works/2011-General-Nonfiction. Accessed March 5, 2013.
  2. University of Texas MD Anderson Center. Moon Shots program. http://cancermoonshots.org/. Accessed March 5, 2013.
  3. Kolata G. In treatment for leukemia, glimpses of the future. New York Times. July 7, 2012.http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?
  4. pagewanted=all&_r=0. Accessed March 5, 2013.



Charles Swanton Publications

London Research Institute

44 Lincoln’s Inn Fields
United Kingdom

WebLab website

Primary research papers

The following publications have been supported by Cancer Research UK funding for this researcher.


From genomic landscapes to personalized cancer management-is there a roadmap?
Swanton C;Caldas C
Ann N Y Acad Sci 2010; 1210 ( ):34-44.
PubMed;  DOI: 10.1111/j.1749-6632.2010.05776.x.

Minimising Immunohistochemical False Negative ER Classification Using a Complementary 23 Gene Expression Signature of ER Status
Li QY;Eklund AC;Juul N;Haibe-Kains B;Workman CT;Richardson AL;Szallasi Z;Swanton C
PLoS ONE 2010; (11):e15031.
DOI: 10.1371/journal.pone.0015031.

How Darwinian models inform therapeutic failure initiated by clonal heterogeneity in cancer medicine
Gerlinger M;Swanton C
Br J Cancer 2010; 103 (8):1139-1143.
UKPubMed (open access);  PubMed;  DOI: 10.1038/sj.bjc.6605912.

Anti-cancer drug resistance: Understanding the mechanisms through the use of integrative genomics and functional RNA interference
Tan DSW;Gerlinger M;Teh BT;Swanton C
Eur J Cancer 2010; 46 (12):2166-2177.
PubMed;  DOI: 10.1016/j.ejca.2010.03.019.

A retrospective analysis of clinical outcome of patients with chemo-refractory metastatic breast cancer treated in a single institution phase I unit
Brunetto AT;Sarker D;Papadatos-Pastos D;Fehrmann R;Kaye SB;Johnston S;Allen M;De Bono JS;Swanton C
Br J Cancer 2010; 103 (5):607-612.
PubMed;  DOI: 10.1038/sj.bjc.6605812.

FKBPL Regulates Estrogen Receptor Signaling and Determines Response to Endocrine Therapy
McKeen HD;Byrne C;Jithesh PV;Donley C;Valentine A;Yakkundi A;O’Rourke M;Swanton C;McCarthy HO;Hirst DG;Robson T
Cancer Res 2010; 70 (3):1090-1100.
DOI: 10.1158/0008-5472.CAN-09-2515.

Prognostic and Predictive Biomarkers in Resected Colon Cancer: Current Status and Future Perspectives for Integrating Genomics into Biomarker Discovery
Tejpar S;Bertagnolli M;Bosman F;Lenz HJ;Garraway L;Waldman F;Warren R;Bild A;Collins-Brennan D;Hahn H;Harkin DP;Kennedy R;Ilyas M;Morreau H;Proutski V;Swanton C;Tomlinson I;Delorenzi M;Fiocca R;Van Cutsem E;Roth A
Oncologist 2010; 15 (4):390-404.
DOI: 10.1634/theoncologist.2009-0233.

Assessment of an RNA interference screen-derived mitotic and ceramide pathway metagene as a predictor of response to neoadjuvant paclitaxel for primary triple-negative breast cancer: a retrospective analysis of five clinical trials
Juul N;Szallasi Z;Eklund AC;Li QY;Burrell RA;Gerlinger M;Valero V;Andreopoulou E;Esteva FJ;Symmans WF;Desmedt C;Haibe-Kains B;Sotiriou C;Pusztai L;Swanton C
Lancet Oncol 2010; 11 (4):358-365.
PubMed;  DOI: 10.1016/S1470-2045(10)70018-8.


RNAi-mediated functional analysis of pathways influencing cancer cell drug resistance
Lee AJX;Kolesnick R;Swanton C
Expert Rev Mol Med 2009; 11 ():e15.
PubMed;  DOI: 10.1017/S1462399409001070.

Advances in personalized therapeutics in non-small cell lung cancer: 4q12 amplification, PDGFRA oncogene addiction and sunitinib sensitivity
Swanton C;Burrell RA
Cancer Biol Ther 2009; (21):2051-2053.

Chromosomal instability A composite phenotype that influences sensitivity to chemotherapy
McClelland SE;Burrell RA;Swanton C
Cell Cycle 2009; (20):3262-3266.

Genetic prognostic and predictive markers in colorectal cancer
Walther A;Johnstone E;Swanton C;Midgley R;Tomlinson I;Kerr D
Nat Rev Cancer 2009; (7):489-499.

Chromosomal instability determines taxane response
Swanton C;Nicke B;Schuett M;Eklund AC;Ng C;Li QY;Hardcastle T;Lee A;Roy R;East P;Kschischo M;Endesfelder D;Wylie P;Kim SN;Chen JG;Howell M;Ried T;Habermann JK;Auer G;Brenton JD;Szallasi Z;Downward J
Proc Natl Acad Sci U S A 2009; 106 (21):8671-8676.
UKPubMed (open access);  PubMed;  DOI: 10.1073/pnas.0811835106.

Molecular classification of solid tumours: towards pathway-driven therapeutics
Swanton C;Caldas C
Br J Cancer 2009; 100 (10):1517-1522.


Epothilones and new analogues of the microtubule modulators in taxane-resistant disease
Harrison M;Swanton C
Expert Opin Invest Drugs 2008; 17 (4):523-546.

Targeting Polo-Like Kinase: Learning Too Little Too Late?
Olmos D;Swanton C;de Bono J
J Clin Oncol 2008; 26 (34):5497-5499.

Concordance of exon array and real-time PCR assessment of gene expression following cancer cell cytotoxic drug exposure
Lee AJX;East P;Pepper S;Nicke B;Szallasi Z;Eklund AC;Downward J;Swanton C
Cell Cycle 2008; (24):3947-3948.

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer
Swanton C;Szallasi Z;Brenton JD;Downward J
Breast Cancer Res 2008; 10 (5):214.
UKPubMed (open access);  PubMed;  DOI: 10.1186/bcr2159.

Unraveling the complexity of endocrine resistance in breast cancer by functional genomics
Swanton C;Downward J
Cancer Cell 2008; 13 (2):83-85.


146 Publications on PubMed by Polyak’s Lab


Other related articles on this Open Access Online Scientific Journal include the following:

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

Stephen J. Williams, Ph.D. 4/10/2013


Pfizer’s Kidney Cancer Drug Sutent Effectively caused REMISSION to Adult Acute Lymphoblastic Leukemia (ALL)

Aviva Lev-Ari, PhD, RN, 7/10/2012


On Tumor and mutations


On ‘genomics mutations’


On ‘cancer sequencing’


 On Metastasis

Read Full Post »

microRNA Biomarker

Reporter: Larry H Bernstein, MD, FCAP

MicroRNA Molecule May Serve as Biomarker

miRNA molecule called miR-7 decreased in highly metastatic cancer stem-like cells.
February 18, 2013
Researchers have identified two molecules that could potentially serve as biomarkers in

MicroRNAs are involved in

  • tumor initiation and
  • progression, and
  • may play a role in metastasis, particularly in relation to
  • cancer stem-like cells.
miR-7 is a metastasis

  • suppressor in cancer stem-like cells, and when they
  • increased expression of miR-7 in cancer stem-like cells from
    • it suppressed their metastatic properties.

miR-7 suppressed ………….expression of KLF4.
However, miR-7 significantly suppressed the ability of cancer stem-like cells to metastasize to the brain but not the bone.

A gram illustrating the disctinction between c...

A gram illustrating the disctinction between cancer stem cell targeted (above) and conventional (below) cancer therapies (Photo credit: Wikipedia)

Related articles


Read Full Post »

Recurrent breast cancer

Recurrent breast cancer (Photo credit: Wikipedia)

Reporter: Venkat Karra, Ph.D. 

There is no good treatmnet for triple-negative breast cancer cells. The standard of care is combination chemotherapy, and although it has a good initial response rate, a significant number of patients develop recurrent cancer,” says Yaffe, who is a member of the David H. Koch Institute for Integrative Cancer Research at MIT.

Yaffe and postdoc Michael Lee, lead author of the Cell paper, focused their study on a type of breast cancer cells known as triple negative, meaning that they don’t have overactive estrogen, progesterone or HER2 receptors. Triple-negative tumors, which account for about 16 percent of breast cancer cases, are much more aggressive than other types and tend to strike younger women.

In the new paper, published in Cell on May 11, the researchers showed that staggering the doses of two specific drugs dramatically boosts their ability to kill a particularly malignant type of breast cancer cells.

Of all combinations they tried, they saw the best results with pretreatment using erlotinib followed by doxorubicin, a common chemotherapy agent. The researchers found that giving erlotinib between four and 48 hours before doxorubicin dramatically increased cancer-cell death. Staggered doses killed up to 50 percent of triple-negative cells, while simultaneous administration killed about 20 percent. About 2,000 genes were affected by pretreatment with erlotinib, the researchers found, resulting in the shutdown of pathways involved in uncontrolled growth.

Here the catch is the ‘order’ and ‘time’ because if the drugs were given in the reverse order, doxorubicin became less effective than if given alone.

They also saw good results with erlotinib and doxorubicin in some types of lung cancer.

“The drugs are going to be different for each cancer case, but the concept that time-staggered inhibition will be a strong determinant of efficacy has been universally true. It’s just a matter of finding the right combinations,” Lee says.

The findings also highlight the importance of systems biology in studying cancer, Yaffe says. “Our findings illustrate how systems engineering approaches to cell signaling can have large potential impact on disease treatment,” he says.


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