Mona L. Gauthier

Biomarker Expression and Risk of Subsequent Tumors After Initial Ductal Carcinoma In Situ Diagnosis

BACKGROUND Studies have failed to identify characteristics of women who have been diagnosed with ductal carcinoma in situ (DCIS) and have a high or low risk of subsequent invasive cancer. METHODS We conducted a nested case-control study in a population-based cohort of 1162 women who were diagnosed with DCIS and treated by lumpectomy alone from 1983 to 1994. We collected clinical characteristics and information on subsequent tumors, defined as invasive breast cancer or DCIS diagnosed in the ipsilateral breast containing the initial DCIS lesion or at a regional or distant site greater than 6 months after initial treatment of DCIS (N = 324). We also conducted standardized pathology reviews and immunohistochemical staining for the estrogen receptor (ER), progesterone receptor, Ki67 antigen, p53, p16, epidermal growth factor receptor-2 (ERBB2, HER2/neu oncoprotein), and cyclooxygenase-2 (COX-2) on the initial paraffin-embedded DCIS tissue. Competing risk models were used to determine factors associated with risk of subsequent invasive cancer vs DCIS, and cumulative incidence survival functions were used to estimate 8-year risk. RESULTS Factors associated with subsequent invasive cancer differed from those associated with subsequent DCIS. Eight-year risk of subsequent invasive cancer was statistically significantly (P = .018) higher for women with initial DCIS lesions that were detected by palpation or that were p16, COX-2, and Ki67 triple positive (p16(+)COX-2(+)Ki67(+)) (19.6%, 95% confidence interval [CI] = 18.0% to 21.3%) than for women with initial lesions that were detected by mammography and were p16, COX-2, and Ki67 triple negative (p16(-)COX-2(-)Ki67(-)) (4.1%, 95% CI = 3.4% to 5.0%). In a multivariable model, DCIS lesions that were p16(+)COX-2(+)Ki67(+) or those detected by palpation were statistically significantly associated with subsequent invasive cancer, but nuclear grade was not. Eight-year risk of subsequent DCIS was highest for women with DCIS lesions that had disease-free margins of 1 mm or greater combined with either ER(-)ERBB2(+)Ki67(+) or p16(+)COX-2(-)Ki67(+) status (23.6%, 95% CI = 18.1% to 34.0%). CONCLUSION Biomarkers can identify which women who were initially diagnosed with DCIS are at high or low risk of subsequent invasive cancer, whereas histopathology information cannot.

Histologically normal human mammary epithelia with silenced p16INK4a overexpress COX-2, promoting a premalignant program

Breast tissue from healthy women contains variant mammary epithelial cells (vHMEC) exhibiting p16INK4a promoter hypermethylation both in vivo and in vitro. When continuously cultured, vHMEC acquire telomeric dysfunction and produce the types of chromosomal abnormalities seen in premalignant lesions of cancer. We find that late passage vHMEC express elevated prostaglandin cyclo-oxygenase 2 (COX-2), which contributes to increased prostaglandin synthesis, angiogenic activity, and invasive ability. These data demonstrate the existence of human mammary epithelial cells with the potential to acquire multiple genomic alterations and phenotypes associated with malignant cells. Moreover, COX-2 overexpression coincides with focal areas of p16INK4a hypermethylation in vivo, creating ideal candidates as precursors to breast cancer. These putative precursors can be selectively eliminated upon exposure to COX-2 inhibitors in vitro.

Genetic and epigenetic changes in mammary epithelial cells may mimic early events in carcinogenesis.

Studies of human mammary epithelial cells from healthy individuals are providing novel insights into how early epigenetic and genetic events affect genomic integrity and fuel carcinogenesis. Key epigenetic changes, such as the hypermethylation of the p16INK4a promoter sequences, create a previously unappreciated preclonal phase of tumorigenesis in which a subpopulation of mammary epithelial cells are positioned for progression to malignancy (Romanov et al., 2001, Nature, 409:633ndash;637; Tlsty et al., 2001, J. Mammary Gland Biol. Neoplasia, 6:235–243). These key changes precede the clonal outgrowth of premalignant lesions and occur frequently in healthy, disease-free women. Understanding more about these early events should provide novel molecular candidates for prevention and therapy of breast cancer that target the process instead of the consequences of genomic instability. This review will highlight some of the key alterations that have been studied in human mammary epithelial cells in culture and relate them to events observed in vivo and discussed in accompanying reviews in this volume.

CD36 Repression Activates a Multicellular Stromal Program Shared by High Mammographic Density and Tumor Tissues

UNLABELLED Although high mammographic density is considered one of the strongest risk factors for invasive breast cancer, the genes involved in modulating this clinical feature are unknown. Tissues of high mammographic density share key histologic features with stromal components within malignant lesions of tumor tissues, specifically low adipocyte and high extracellular matrix (ECM) content. We show that CD36, a transmembrane receptor that coordinately modulates multiple protumorigenic phenotypes, including adipocyte differentiation, angiogenesis, cell-ECM interactions, and immune signaling, is greatly repressed in multiple cell types of disease-free stroma associated with high mammographic density and tumor stroma. Using both in vitro and in vivo assays, we show that CD36 repression is necessary and sufficient to recapitulate the above-mentioned phenotypes observed in high mammographic density and tumor tissues. Consistent with a functional role for this coordinated program in tumorigenesis, we observe that clinical outcomes are strongly associated with CD36 expression. SIGNIFICANCE CD36 simultaneously controls adipocyte content and matrix accumulation and is coordinately repressed in multiple cell types within tumor and high mammographic density stroma, suggesting that activation of this stromal program is an early event in tumorigenesis. Levels of CD36 and extent of mammographic density are both modifiable factors that provide potential for intervention.

p38 Regulates Cyclooxygenase-2 in Human Mammary Epithelial Cells and Is Activated in Premalignant Tissue

The immediate-early gene, cyclooxygenase-2 (COX-2), is induced in a variety of inflammatory and neoplastic processes and is believed to play an important role in tumorigenesis. In this study, we identify an important upstream regulatory pathway of COX-2 expression in variant human mammary epithelial cells (vHMEC), which has been shown to exhibit phenotypes important for malignancy. We find that the stress-activated kinase, p38, is phosphorylated and activated in vHMEC compared with HMEC and is responsible for the expression of COX-2 in vHMEC as cells grow in culture. Furthermore in this capacity, p38 acts to stabilize the COX-2 transcript rather than activate COX-2 transcription. Inhibition of p38 kinase, using a chemical inhibitor, down-regulates COX-2 and decreases cell survival. Examination of archived tissue from women with ductal carcinoma in situ reveals epithelial cells that not only overexpress COX-2 but also have an abundance of activated phospho-p38 in the nucleus and cytoplasm, mirroring the expression observed in vitro. These epithelial cells are found within premalignant lesions as well as in fields of morphologically normal tissue that surround the lesions. In contrast, low phospho-p38 staining was observed in the majority of normal tissue obtained from reduction mammoplasty. These data help define the regulation of COX-2 expression in early carcinogenesis and provide alternative candidates for targeted prevention of COX-2-induced phenotypes and breast cancer.

Matrix Metalloproteinase Induction of Rac1b, a Key Effector of Lung Cancer Progression

Rac1b mediates the matrix metalloproteinase–induced epithelial-mesenchymal transition in lung adenocarcinoma and is a potential therapeutic target in early-stage lung cancer. Neutralizing the Neighbors Life-long addictions to cigarettes—and the accompanying enhanced risk of lung cancer—often begin with a succumbing to peer pressure. Now, Stallings-Mann et al. characterize lung cancer–related peer pressure at the molecule level by showing that a protease in the extracellular matrix compels expression, in neighboring lung cells, of a signaling protein (Rac1B) that acts as a mediator of lung cancer progression. Exposure to cigarette smoke is the key cause of non–small cell lung cancers (NSCLCs), which constitute >80% of all cases of lung cancer. NSCLC is most effectively treated when detected early, but to stop this cancer in its tracks, scientists must learn more about the molecular mechanisms of tumor progression and then use this information to discover new targeted therapies. Rac1b is a tumor-associated, cell-transforming protein that arises as an alternatively spliced isoform of Rac1, a Rho family GTPase that regulates cell proliferation. It has been hypothesized that Rac1b drives oncogenesis by promoting epithelial-mesenchymal transition (EMT), during which epithelial cells detach from each other and from the underlying basement membrane and acquire invasive properties. Matrix metalloproteinases (MMPs) live in and cleave components of the extracellular matrix that borders epithelial cells and have been shown to induce EMT. Stallings-Mann et al. found that expression of mouse Rac1b in lung epithelial cells of transgenic mice spurred both EMT and spontaneous tumor formation. Furthermore, in the transgenic mice, MMP3-induced expression of Rac1b stimulated EMT and progression of the premalignant state in lung epithelial cells to malignant lung adenocarcinoma by bypassing oncogene-induced senescence. In patients, Rac1b mRNA expression was elevated in lung adenocarcinoma relative to adjacent normal tissue, in stage-2 relative to stage-1 lung tumors, and in normal tissue from smokers compared to nonsmokers. How MMP3, Rac1b, and EMT collaborate to drive tumor progression remains unclear. But the new work suggests that drugs that block synthesis or function of Rac1b—which has no known function in normal cells—may prevent progression to late-stage, invasive forms of lung cancer. Lung cancer is more deadly than colon, breast, and prostate cancers combined, and treatment improvements have failed to improve prognosis significantly. Here, we identify a critical mediator of lung cancer progression, Rac1b, a tumor-associated protein with cell-transforming properties that are linked to the matrix metalloproteinase (MMP)–induced epithelial-mesenchymal transition (EMT) in lung epithelial cells. We show that expression of mouse Rac1b in lung epithelial cells of transgenic mice stimulated EMT and spontaneous tumor development and that activation of EMT by MMP-induced expression of Rac1b gave rise to lung adenocarcinoma in the transgenic mice through bypassing oncogene-induced senescence. Rac1b is expressed abundantly in stages 1 and 2 of human lung adenocarcinomas and, hence, is an attractive molecular target for the development of new therapies that prevent progression to later-stage lung cancers.

p16INK4a modulates p53 in primary human mammary epithelial cells.

p16(INK4a) (p16) and p53 are tumor suppressor genes that are inactivated during carcinogenesis in many tumors. Here we show that p16 gene activity inversely modulates p53 status and function in primary human mammary epithelial cells. Reduced levels of p16 protein stabilize p53 protein through inhibition of proteolytic degradation, and this increase in p53 protein levels enhances the cellular response to radiation, represses proliferation, and transcriptionally activates downstream targets. Stabilization of p53 is mediated through the retinoblastoma/E2F/p14(ARF)/murine double minute-2 pathway. However, we have observed that p16 does not modulate p53 in fibroblasts, indicating a possible cell type-specific regulation of this pathway.

Estrogen controls the survival of BRCA1-deficient cells via a PI3K–NRF2-regulated pathway

Significance Our establishment of a connection between the phosphatidylinositol 3-kinase (PI3K) and NRF2 pathways provides the basis for the tissue specificity of BRCA1-related cancers. Because BRCA1 is a vital component of the intracellular machinery maintaining genomic stability, BRCA1 functions as a major tumor suppressor in cells of all types. However, BRCA1-related cancers occur overwhelmingly in breasts and ovaries. Our work demonstrates that estrogen (E2) acts via the PI3K–AKT pathway to regulate NRF2 activation in BRCA1-deficient cells, resulting in the induction of antioxidant genes that protect the cell from reactive oxygen species-induced death. BRCA1-deficient cells are thus allowed to survive and may accumulate mutations, such as loss of PTEN, that perpetuate NRF2 activation. Our work supports the emerging clinical strategy of treating BRCA1-related cancers with PI3K inhibitors. Mutations in the tumor suppressor BRCA1 predispose women to breast and ovarian cancers. The mechanism underlying the tissue-specific nature of BRCA1’s tumor suppression is obscure. We previously showed that the antioxidant pathway regulated by the transcription factor NRF2 is defective in BRCA1-deficient cells. Reactivation of NRF2 through silencing of its negative regulator KEAP1 permitted the survival of BRCA1-null cells. Here we show that estrogen (E2) increases the expression of NRF2-dependent antioxidant genes in various E2-responsive cell types. Like NRF2 accumulation triggered by oxidative stress, E2-induced NRF2 accumulation depends on phosphatidylinositol 3-kinase–AKT activation. Pretreatment of mammary epithelial cells (MECs) with the phosphatidylinositol 3-kinase inhibitor BKM120 abolishes the capacity of E2 to increase NRF2 protein and transcriptional activity. In vivo the survival defect of BRCA1-deficient MECs is rescued by the rise in E2 levels associated with pregnancy. Furthermore, exogenous E2 administration stimulates the growth of BRCA1-deficient mammary tumors in the fat pads of male mice. Our work elucidates the basis of the tissue specificity of BRCA1-related tumor predisposition, and explains why oophorectomy significantly reduces breast cancer risk and recurrence in women carrying BRCA1 mutations.

Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF.

Purpose: p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer. Experimental Design: To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index. Results:INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-β2, estrogen receptor-α, and breast cancer-associated 1 genes (P = 0.001). Conclusions: Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.

Human mammary cancer progression model recapitulates methylation events associated with breast premalignancy

IntroductionWe have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.MethodsHMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).ResultsvHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.ConclusionsWe have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.