Gene In Human Breast Cancer Research Articles

Abstract 443: Expression of the RECK tumor and metastasis suppressor gene in human breast cancer: a poor prognosis marker

Abstract Metastasis is the main cause of mortality in breast cancer human patients. The organized breakdown of the extracellular matrix (ECM) compounds by matrix metalloproteinases (MMPs) is the key event in this metastatic process. Consistent with their role in tumor progression, high levels of several MMP family members have been shown to be correlated with poor prognosis. The MMP activities are tightly regulated by their inhibitors, namely: the metalloproteinases tissue inhibitors (TIMPs) and the membrane-associated inhibitor (RECK - Reversion-inducing Cysteine-rich protein with Kazal motifs). Due to RECK's ability to down modulate MMPs (MMP-2, 9 and 14), it is described as an important inhibitor of invasion and metastasis in several types of cancer. In order to investigate possible correlations between the RECK expression profile and clinical-pathological data in a breast cancer model, we evaluated the RECK protein in 1,040 tissue samples from invasive breast tumors using Tissue Microarray Assays (TMA). Associations between RECK expression and different clinical-pathological parameters and other biomarkers were evaluated using the Chi-square test. RECK immunoreactivity was significantly associated with higher SBR grade (p<0.05) and TNM stage (p=0.002). Moreover, higher RECK expression levels were correlated with p53 (p<0.001), her-2 (p=0.011), CK5,6 (p<0.001) and egfr (p<0.001) positivity. We also analyzed the relationship between RECK expression and the patient outcome, using Kaplan-Meier curves, demonstrating that patients with RECK positive tumors display significantly shorter disease free survival time (p=0.031) than those with RECK negative tumors. Furthermore, RECK expression was positively correlated with short disease free survival in five-years in patients diagnosed with tumors: low SBR grade (p=0.03), her-2 negative (p<0.05) and luminal markers (CK8/CK18) positive (p=0.02 and p=0.003, respectively). In addition, RECK was a predictor of poor prognosis for post-menopause (p=0.011) and patients older than 50 years (p=0.05). All together, our results indicate that higher RECK expression is associated with poor prognosis variables, being a predictor of shorter disease-free survival in five years. Moreover, we verified that RECK is a biomarker of poor prognosis mainly for patients diagnosed with less aggressive breast tumor. Therefore, in contrast to other tumor types, our results indicate that RECK is related to a more aggressive phenotype. Support: FAPESP, CNPq, FINEP, CAPES, BNDES Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 443. doi:1538-7445.AM2012-443

P3-17-01: ApoE and Its Receptors (LRP8, VLDLR) Function as Growth Signals for Triple-Negative Breast Cancer and Represent a Novel Therapeutic Target.

Abstract Triple-negative breast cancers (TNBC) lack the expression of estrogen, progesterone, and the human epidermal growth factor 2 receptors and have limited treatment options. We hypothesized that genes that are frequently amplified or overexpressed in these cancers are functionally important for the growth and survival of triple-negative breast cancer. Materials/Methods: We identified genes overexpressed in TNBC in 3 different human breast cancer gene expression data sets (n=294, n=286, n=198). We assessed the functional importance of the consistently overexpressed genes using siRNA screen on 18 breast cancer cell lines (10 ER-, 8 ER+) in vitro. We targeted each gene with 4 different siRNA constructs separately and each experiment was performed in triplicate. The genes with the greatest TNBC-specific inhibitory effect after siRNA down regulation were selected for further mechanistic and signal transduction studies. Results: 684 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in all 3 data sets. siRNA suppression of 161 of these genes inhibited cell growth significantly more in the ER-negative compared to ER-positive cells by at least 1 of the 4 siRNAs, 27 genes showed similar effect with 2 or more siRNAs and for 2 genes 3 of the 4 siRNAs showed preferential growth inhibition in ER-negative cells. These two genes were VLDLR (very low-density lipoprotein receptor) and LRP8 (low density lipoprotein receptor-related protein 8). We validated the siRNA screen results and confirmed down regulation of the mRNA and protein levels for VLDLR and LRP8 in 4 different ER-negative cell lines and showed that siRNA inhibition can be rescued by co-transfection of the receptor genes. Reanalysis of gene expression data also indicated expression of the LRP8 and VLDLR ligands, Reelin or ApoE, in both breast cancer tissues and in cell lines. We next demonstrated that exposure to Reelin and ApoE stimulates the growth of ER-negative cells in vitro. The stimulatory effect of ApoE was isoform dependent ApoE2 (Cys112/Cys158;) has the lowest receptor binding affinity and showed no growth stimulation, ApoE3 (Cys112/ Arg158) had modest 50–60% growth stimulation and ApoE4 (Arg112/ Arg158) had the greatest stimulatory effect (300-400%). Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. ApoE4 stimulation results in the transcriptional upregulation of genes involved in proliferation, metabolism, and inflammatory signaling pathways. ApoE4 stimulation increases expression of proteins involved in MAPK/ERK pathway, DNA damage repair, and inflammation. Conclusions: We show that theApoE - LRP8/VLDLR ligand receptor system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro. Interestingly, allelic imbalance favoring ApoE4 expression (E3/E4 or E4/E4) has been linked to higher risk of developing early onset breast cancer, which is primarily TNBC. We propose that inhibitors of LRP8/VLDLR signaling may be clinically useful therapeutic or preventive agents for TNBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-01.

Definition of the Landscape of Promoter DNA Hypomethylation in Liver Cancer

We use hepatic cellular carcinoma (HCC), one of the most common human cancers, as a model to delineate the landscape of promoter hypomethylation in cancer. Using a combination of methylated DNA immunoprecipitation and hybridization with comprehensive promoter arrays, we have identified approximately 3,700 promoters that are hypomethylated in tumor samples. The hypomethylated promoters appeared in clusters across the genome suggesting that a high-level organization underlies the epigenomic changes in cancer. In normal liver, most hypomethylated promoters showed an intermediate level of methylation and expression, however, high-CpG dense promoters showed the most profound increase in gene expression. The demethylated genes are mainly involved in cell growth, cell adhesion and communication, signal transduction, mobility, and invasion; functions that are essential for cancer progression and metastasis. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine, activated several of the genes that are demethylated and induced in tumors, supporting a causal role for demethylation in activation of these genes. Previous studies suggested that MBD2 was involved in demethylation of specific human breast and prostate cancer genes. Whereas MBD2 depletion in normal liver cells had little or no effect, we found that its depletion in human HCC and adenocarcinoma cells resulted in suppression of cell growth, anchorage-independent growth and invasiveness as well as an increase in promoter methylation and silencing of several of the genes that are hypomethylated in tumors. Taken together, the findings define the potential functional role of hypomethylation in cancer.

Downregulation and antiproliferative role of fhl3 in breast cancer

Four and a half LIM domain (FHL) protein 3 is a member of the FHL protein family that plays roles in the regulation of signal transduction, cell adhesion, survival, and mobility. FHL3 has been implicated in the development and progression of liver cancer. However, the biological function of FHL3 in other cancers remains unclear. Here, we show that FHL3 is downregulated in breast cancer patients. Using small interfering RNA (siRNA) knockdown and/or overexpression experiments, we demonstrated that FHL3 suppressed anchorage-dependent and -independent growth of human breast cancer cells. The antiproliferative effects of FHL3 on breast cancer cell growth were associated with both the G1 and the G2/M cell cycle arrest, which was accompanied by a marked inhibition of the G1-phase marker cyclin D1 and the G2/M-phase marker cyclin B1 as well as the induction of the cyclin dependent kinase inhibitor p21 (WAF1/CIP1), a negative regulator of cell cycle progression at G1 and G2. These results suggest that FHL3 may play a role in the development and progression of breast cancer, and thereby may be a potential target for human breast cancer gene therapy.

Induction of Stable Drug Resistance in Human Breast Cancer Cells Using a Combinatorial Zinc Finger Transcription Factor Library

Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust tool for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to fulvestrant, a clinically important anti-endocrine therapeutic agent. From a diverse collection of nearly 400,000 different ZF-TFs, we isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor-positive breast cancer cell lines. Comparative gene expression profile analysis of the six different ZF-TF-transduced breast cancer cell lines revealed five distinct clusters of differentially expressed genes. One cluster was shared among all 6 ZF-TF-transduced cell lines and therefore constituted a common fulvestrant-resistant gene expression signature. Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents. Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes. Finally, we also demonstrated that the common fulvestrant-resistant signature is associated with poor prognosis by interrogating five independent, publicly available human breast cancer gene expression datasets. Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

Distinct expressions of microRNAs that directly target estrogen receptor α in human breast cancer

Estrogen receptor (ER) α is essential for estrogen-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ERα-positive breast cancer. Breast cancer patients show a wide range of ERα expression levels and the levels of expression in individual patients change during disease progression and in response to systemic therapies. However, little is known about how the expression of ERα in human breast cancer is regulated. Recently, several microRNAs (miRNAs) that directly target ERα have been identified, and we previously demonstrated that miR-206 expression was downregulated in ERα-positive human breast cancer. In this study, expression levels of miRNAs that directly target ERα, including miR-18a, miR-18b, miR-22, miR-193b, miR-221/222 and miR-302c, were analyzed in human breast cancer samples by quantitative reverse transcription-PCR analysis. Correlations between the expression levels of these miRNAs and clinicopathological factors, including prognosis, were analyzed. miR-18a expression was much higher in ERα-negative than in ERα-positive tumors (P < 0.0001), with the expression levels of miR-18a not differing in ERα-positive breast cancer as a function of ERα protein level. Surprisingly, the expression levels of miR-193b and miR-221 were significantly lower in ERα-negative than in ERα-positive tumors (P = 0.0015 and P = 0.0045, respectively), and the levels of these miRNAs gradually increased as ERα protein expression increased. There was no statistically significant association between miR-22 and ERα expression, and miR-302c expression was minimal in human breast cancer samples. Prognostic analysis showed that low miR-18b expression was significantly associated with improved survival in HER2-negative breast cancer, although miR-18b expression was not correlated with ERα protein expression. Our results suggest that miRNAs that directly target ERα have distinct roles in not only regulating ERα but also regulating other target genes in human breast cancer.

Transcriptional down‐regulation of Brca1 and E‐cadherin by CtBP1 in breast cancer

Carboxyl-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor with oncogenic potential. Immunohistochemistry staining using human breast cancer tissue arrays revealed that 92% of invasive ductal breast cancer cases have CtBP1-positive staining compared to 4% CtBP1-positive in normal breast tissue. To explore the functional impact of CtBP1 in breast cancer, we examined CtBP1's transcriptional regulation of known tumor suppressors, breast cancer susceptibility gene 1 (Brca1), and E-cadherin. We found CtBP1 was recruited to the promoter regions of Brca1 and E-cadherin genes in breast cancer cells. Concomitantly, Brca1 loss was detected in 57% and E-cadherin loss was detected in 76% of human invasive ductal breast cancers, and correlated with CtBP1 nuclear staining in these lesions. Importantly, siRNA knock down of CtBP1 restored Brca1 and E-cadherin expression in breast cancer cell lines, implying CtBP1 down-regulates Brca1 and E-cadherin genes in human breast cancer. This study provides evidence that although genetic loss of Brca1 and E-cadherin are infrequent in breast cancer, they are down-regulated at the transcriptional level by CtBP1 expression. Thus, CtBP1 activation could be a potential biomarker for breast cancer development.

Identification and functional analysis of 9p24 amplified genes in human breast cancer

Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16(INK4a), p21(Waf1/Cip1) and p27(Kip1). Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.

Abstract 4700: Identification and functional analysis of 9p23-24 amplified genes in human breast cancer

Abstract Gene amplification is a major mechanism used by cancer cells to increase gene expression and subsequently gain a growth and survival advantage. Previously, we identified a novel amplicon at 9p23-24 region in human esophageal and breast cancer and originally cloned a novel gene GASC1 (Gene Amplified in Squamous Cell Carcinoma 1) from this amplicon. Accumulating evidence suggests common amplicons that occur in breast and other cancers contain multiple oncogenes that could play a role in cancer initiation and progression. In the current study, we aimed to comprehensively characterize the 9p23-24 amplified genes in human breast cancer. We performed array CGH analysis on a panel of cancer cell lines with the Agilent 244K microarray chip and narrowed the shortest region of overlapping (SRO) of the 9p23-24 amplicon to ∼2 Mb. Eleven genes within the SRO regions were examined for their mRNA expression by quantitative RT-PCR (qRT-PCR). Based on statistical association between copy number and expression, we confirmed GASC1 as a top candidate oncogene and identified three new potential oncogenes, UHRF2, KIAA1432 and C9orf123. Our more recent studies indicated that GASC1 is a member of a new class of oncogenes that are involved in the deregulation of histone methylation in cancer cells. We found that GASC1 induces the expression of core stemness transcription factors NANOG, SOX2 and OCT4, as well as classical oncogenes, including NOTCH1 and MYC, in breast cancer cells. On the other hand, UHRF2 encodes a nuclear protein that is involved in cell-cycle regulation. Knocking down UHRF2 slowed cell growth and inhibited colony formation of 9p23-24 amplified breast cancer cells. Furthermore, we demonstrated that expression levels of key tumor suppressing genes, including p16/INK4a, p21/WAF1 and p27/KIP1, were increased after knocking down UHRF2 in breast cancer cells. Collectively, our studies support the notion that the 9p23-24 amplicon contains multiple candidate oncogenes and may play an important role in human tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4700. doi:10.1158/1538-7445.AM2011-4700

Abstract 3097: Global transcript profiling for prolactin-stat5 dependent target genes in breast cancer

Abstract Transcription factor Stat5 is a key mediator of prolactin receptor signaling and plays important roles in both normal mammary development and breast cancer. Although numerous studies support tumor-promoting roles for Stat5, accumulating evidence also points to a role of Stat5 in promoting cell differentiation in breast cancer cells. In fact, high levels of tyrosine phosphorylated Stat5 (pY-Stat5) in human breast cancer specimens are correlated with favorable prognosis. Identifying Stat5 target genes in human breast cancer may shed new light on the roles of Stat5 as well as provide new candidate prognostic biomarkers. This study was designed to identify candidate Stat5 target genes using tumor extracts of the breast cancer cell line T47D xenotransplanted into nude mice that were subsequently treated with or without human prolactin. Global transcript profiling on the Affymetrix U133 chip platform was employed. The analysis revealed 97 genes that were significantly modulated by prolactin, based on fold difference greater than 1.7 and a p-value less than 0.01. Among these, 61 genes were upregulated and 36 genes were downregulated by prolactin. A total of 18 transcripts were selected based on their relevance to breast cancer and these genes were further investigated using qRT-PCR analysis. Of the 18 candidate targets, 15 were regulated by prolactin in vivo. Of these, 13 were also regulated by prolactin in T47D cells in vitro. Given that prolactin signals through multiple pathways other than Stat5, we examined the ability of Stat5 to regulate the newly identified prolactin-dependent genes by over-expressing Stat5a or Stat5b in the T47D cells. Twelve of the 13 genes tested responded by increased degree of prolactin-modulation following ectopic over-expression of Stat5a or Stat5b, supporting the notion that these genes are downstream targets of Stat5. The identified candidate Stat5 target genes are known to have diverse functions that may critically impact breast cancer growth and progression. For instance, NTN4 is a transmembrane protein whose expression levels positively correlates with better prognosis in breast cancer. AREG and EREG are known to activate the ErbB receptors, whose functions are essential for breast cancer progression. FN1 is a known factor involved in breast cancer invasion. Lastly, PTHrP is well established as being functionally involved in breast cancer metastasis. Fluorescence-based quantitative immunohistochemistry revealed a highly positive correlation between levels of pY-Stat5 and PTHrP proteins in human clinical breast cancer specimens, providing proof-of-principle that the combination of global transcript profiling and quantitative immunohistochemistry analysis is valuable for identifying potential breast cancer biomarkers. Efforts are underway to examine the function of these and additional Stat5 target genes in order to further elucidate the roles of Stat5 in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3097. doi:10.1158/1538-7445.AM2011-3097

Abstract LB-219: ApoE and its' receptors (LRP8, VLDLR) function as growth signals for triple-negative breast cancer and represents novel therapeutic targets

Abstract Background: Triple-negative breast cancers (TNBC) lack the expression of estrogen, progesterone, and the human epidermal growth factor 2 receptors and have limited treatment options. We hypothesized that genes that are frequently amplified or overexpressed in these cancers are functionally important for the growth and survival of triple-negative breast cancer. Materials/Methods: We identified genes overexpressed in TNBC in 3 different human breast cancer gene expression data sets (n=294, n=286, n=198). We assessed the functional importance of the consistently overexpressed genes using siRNA screen on 18 breast cancer cell lines (10 ER−, 8 ER+). We targeted each gene with 4 different siRNAs separately and each experiment was performed in triplicate. The genes with the greatest TNBC-specific inhibitory effect after siRNA down regulation were selected for further mechanistic studies. Results: 684 genes showed consistent and highly significant overexpression in TNBC compared to receptor-positive cancers in all 3 data sets (11% of these mapped to DNA segments with frequent copy number gains). 27 genes showed similar effect with 2 or more siRNAs and for 2 genes 3 of the 4 siRNAs showed preferential growth inhibition in ER-negative cells. These two genes were VLDLR (very low-density lipoprotein receptor) and LRP8 (low density lipoprotein receptor-related protein 8). We validated the siRNA screen results and confirmed down regulation of the mRNA and protein levels for VLDLR and LRP8 in 4 different ER-negative cell lines and showed that siRNA inhibition can be rescued by co-transfection of the receptor genes. Reanalysis of gene expression data also indicated expression of the LRP8 and VLDLR ligands, Reelin or ApoE, in both breast cancer tissues and in cell lines. We next demonstrated that exposure to Reelin and ApoE stimulates the growth of ER-negative cells in vitro. The stimulatory effect of ApoE was isoform dependent; ApoE2 (Cys112/Cys158) has the lowest receptor binding affinity and showed no growth stimulation, ApoE3 (Cys112/Arg158) had modest 50–60% growth stimulation and ApoE4 (Arg112/Arg158) had the greatest stimulatory effect (300–400%). Suppression of the expression of either LRP8 or VLDLR or exposure to RAP (an inhibitor of ligand binding to LRP8 and VLDLR) abolished this ligand-induced proliferation. Conclusions: We show that the ApoE -LRP8/VLDLR ligand receptor system is overexpressed in human TNBC. We also demonstrated that this receptor system mediates a strong growth promoting and survival function in TNBC cells in vitro. Interestingly, allelic imbalance favoring ApoE4 expression (E3/E4 or E4/E4) has been linked to higher risk of developing early onset breast cancer, that is primarily TNBC. We propose that inhibitors of LRP8/VLDLR signaling may be clinically useful therapeutic agents for TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-219. doi:10.1158/1538-7445.AM2011-LB-219

Seeing the Forest Through the Trees: Gene Patents &amp; the Reality of the Commons

Patents prevent anyone but the patent-holder from manufacturing, using, or distributing discoveries and inventions for twenty years from the date of filing. In order to be patentable, an invention needs to be useful, non-obvious, and represent an original design or process rather than an abstract concept or item commonly found in nature. Patents related to genetics received their first legal test in 1980, when the U.S. Patent and Trademark Office (USPTO) granted protection to a genetically engineered bacterium that consumed oil and was useful in cleaning oil spills. The legality of this patent was affirmed in Diamond v. Chakrabarty, where the Supreme Court observed that although “[t]he laws of nature, physical phenomena, and abstract ideas” were not patentable subject matter under § 101, the claimed invention in the case was distinguished from nature as “a product of human ingenuity having a distinctive name, character and use.” The Court held that although the invention comprised a living thing, the patentee had produced a new bacterium with “markedly different characteristics” from the original. The bacterium was, therefore, “not nature’s handiwork but [the patentee’s] own.” Although Chakrabarty settled the question of whether manufactured genes can receive patent protection, it did not address the patentability of naturally occurring genes. In the absence of such definitive legal guidance, the USPTO routinely issues patents on human deoxyribonucleic acid (DNA) sequences, reasoning that the material has been purified from its natural form through human intervention and is thus sufficiently “touched by man” to be beyond the scope of nature. From 1980 to 2009, the USPTO issued between 3,000 and 5,000 patents on human genes, encompassing nearly 20% of the human genome. In addition, the USPTO has issued nearly 50,000 patents involving human genetic material, yet the fundamental validity of such patents has never been reviewed until now. In March 2010, a district court decision in New York brought attention to the role of gene patents in the advancement of biomedical research. In Association for Molecular Pathology v. United States Patent and Trademark Office (“AMP”), the Southern District of New York enforced a strict standard for subject matter patentability by invalidating seven patents relating to the human breast cancer genes BRCA1 and BRCA2 (collectively “BRCA”). The court reasoned that not only were the coding sequences and mutations of BRCA results of natural phenomena but that, the purified forms of BRCA maintain essentially the same structures and functions as their natural forms and therefore fall outside the scope of patent law protection. Although the decision primarily addressed the patent’s subject matter, the court also noted the possible social implications resulting from how patents affect access and innovation in biomedical research. Contrary to concerns raised by the plaintiffs in AMP, empirical studies indicate that gene patents do not impede access to biomedical research data or play a significant role in influencing the topics of research that scientists choose to pursue. These results suggest that while gene patents do not impede innovation, they may not be necessary for it either, at least at the foundational level. Some scholars still maintain, however, that patent protection is necessary to ensure adequate funding for further research, development, and marketing of their innovations. This Note focuses on the role of patent law in encouraging or discouraging innovation in the field of biomedical research. Specifically, this Note analyzes the policy justifications underlying gene patents and explores whether these justifications validly apply to the patenting of the BRCA gene. Part I establishes a basic understanding of patents, genes, and gene patents. Part II provides greater detail regarding the arguments and holding in the AMP case. Part III introduces the traditional rationales for patent protection and applies them to gene patents. Part IV considers the concerns surrounding gene patents and whether these concerns are realistic given the results of empirical studies on the relationship between patents and biomedical research. Part IV also examines whether the district court’s holding in AMP is consistent with the policy goals behind intellectual property rights and the reality of the industry. Finally, this Note concludes that, in general, patents do not impede upon innovation. However, the broad issuance of composition claims, such as those held by Myriad in AMP, may block research in areas of study that the patent holder is not pursuing (such as therapeutics). This Note suggests that this issue could be resolved by narrowing the focus of the patent claim to the application of the gene composition, rather than the composition on its own.

Cooperation between Pik3ca and p53 Mutations in Mouse Mammary Tumor Formation

PIK3CA, which codes for the p110α catalytic subunit of phosphatidylinositol 3-kinase, is one of the most frequently mutated genes in human breast cancer. Here, we describe a mouse model for PIK3CA-induced breast cancer by using the ROSA26 (R26) knock-in system, in which targeted Pik3ca alleles can be activated through transgenic expression of Cre recombinase. We mated Pik3ca(H1047R) and Pik3ca(wt) knock-in lines with MMTV-Cre transgenics, which express Cre in mammary epithelium. Starting at approximately 5 months of age, female R26-Pik3ca(H1047R);MMTV-Cre mice, but not control R26-Pik3ca(wt);MMTV-Cre mice, developed mammary tumors, as well as lymphoid and skin malignancies. R26-Pik3ca(H1047R);MMTV-Cre mammary tumors were typically either adenosquamous carcinoma or adenomyoepithelioma. As p53 is the most commonly mutated gene in breast cancer, we tested for genetic interaction between Pik3ca(H1047R) and p53 loss-of-function mutations in R26-Pik3ca(H1047R);p53(loxP/+);MMTV-Cre mice. This led to decreased survival of double-mutant animals, which developed lymphoma and mammary tumors with rapid kinetics. Mammary tumors that formed in p53(loxP/+);MMTV-Cre conditional mutants were either poorly differentiated adenocarcinoma or spindle cell/EMT, whereas R26-Pik3ca(H1047R);p53(loxP/+);MMTV-Cre mammary tumors were mostly adenosquamous carcinoma or spindle cell/EMT indicating that double-mutant mice develop a distinct spectrum of mammary tumors. Thus, an oncogenic variant of PIK3CA implicated in multiple human breast cancer subtypes can induce a very diverse spectrum of mammary tumors in mice. Furthermore, Pik3ca(H1047R) shows cooperation with p53, which altered the specific tumors that formed. Thus, the two most frequently mutated genes in human breast cancer show cooperation in mammary tumor formation.

SPA‐1 controls the invasion and metastasis of human prostate cancer

Recent studies suggest that SIPA1 encoding a Rap GTPase-activating protein SPA-1 is a candidate metastasis efficiency-modifying gene in human breast cancer. In this study, we investigated the expression and function of SPA-1 in human prostate cancer (CaP). Immunohistochemical studies of tumor specimens from CaP patients revealed a positive correlation of SPA-1 expression with disease progression and metastasis. The correlation was recapitulated in human CaP cell lines; LNCaP that rarely showed metastasis in SCID mice expressed an undetectable level of SPA-1, whereas highly metastatic PC3 showed abundant SPA-1 expression. Moreover, SIPA1 transduction in LNCaP caused prominent abdominal lymph node metastasis without affecting primary tumor size, whereas shRNA-mediated SIPA1 knockdown or expression of a dominant-active Rap1 mutant (Rap1V12) in PC3 suppressed metastasis. LNCaP transduced with SPA-1 (LNCaP/SPA-1) showed attenuated adhesion to the precoated extracellular matrices (ECM) including collagens and fibronectin, due to defective ECM-medicated Rap1 activation. In addition, LNCaP/SPA-1 showed a diminished level of nuclear Brd4, which is known to bind SPA-1, resulting in reduced expression of a series of ECM-related genes. These results suggest that SPA-1 plays an important role in controlling metastasis efficiency of human CaP by regulating the expression of and interaction with ECM in the primary sites.

Seeing the Forest through the Trees: Gene Patents & the Reality of the Commons

Patents prevent anyone but the patent-holder from manufacturing, using, or distributing discoveries and inventions for twenty years from the date of filing. In order to be patentable, an invention needs to be useful, non-obvious, and represent an original design or process rather than an abstract concept or item commonly found in nature. Patents related to genetics received their first legal test in 1980, when the U.S. Patent and Trademark Office (USPTO) granted protection to a genetically engineered bacterium that consumed oil and was useful in cleaning oil spills. The legality of this patent was affirmed in Diamond v. Chakrabarty, where the Supreme Court observed that although “[t]he laws of nature, physical phenomena, and abstract ideas” were not patentable subject matter under § 101, the claimed invention in the case was distinguished from nature as “a product of human ingenuity having a distinctive name, character and use.” The Court held that although the invention comprised a living thing, the patentee had produced a new bacterium with “markedly different characteristics” from the original. The bacterium was, therefore, “not nature’s handiwork but [the patentee’s] own.” Although Chakrabarty settled the question of whether manufactured genes can receive patent protection, it did not address the patentability of naturally occurring genes. In the absence of such definitive legal guidance, the USPTO routinely issues patents on human deoxyribonucleic acid (DNA) sequences, reasoning that the material has been purified from its natural form through human intervention and is thus sufficiently “touched by man” to be beyond the scope of nature. From 1980 to 2009, the USPTO issued between 3,000 and 5,000 patents on human genes, encompassing nearly 20% of the human genome. In addition, the USPTO has issued nearly 50,000 patents involving human genetic material, yet the fundamental validity of such patents has never been reviewed until now. In March 2010, a district court decision in New York brought attention to the role of gene patents in the advancement of biomedical research. In Association for Molecular Pathology v. United States Patent and Trademark Office (“AMP”), the Southern District of New York enforced a strict standard for subject matter patentability by invalidating seven patents relating to the human breast cancer genes BRCA1 and BRCA2 (collectively “BRCA”). The court reasoned that not only were the coding sequences and mutations of BRCA results of natural phenomena but that, the purified forms of BRCA maintain essentially the same structures and functions as their natural forms and therefore fall outside the scope of patent law protection. Although the decision primarily addressed the patent’s subject matter, the court also noted the possible social implications resulting from how patents affect access and innovation in biomedical research. Contrary to concerns raised by the plaintiffs in AMP, empirical studies indicate that gene patents do not impede access to biomedical research data or play a significant role in influencing the topics of research that scientists choose to pursue. These results suggest that while gene patents do not impede innovation, they may not be necessary for it either, at least at the foundational level. Some scholars still maintain, however, that patent protection is necessary to ensure adequate funding for further research, development, and marketing of their innovations. This Note focuses on the role of patent law in encouraging or discouraging innovation in the field of biomedical research. Specifically, this Note analyzes the policy justifications underlying gene patents and explores whether these justifications validly apply to the patenting of the BRCA gene. Part I establishes a basic understanding of patents, genes, and gene patents. Part II provides greater detail regarding the arguments and holding in the AMP case. Part III introduces the traditional rationales for patent protection and applies them to gene patents. Part IV considers the concerns surrounding gene patents and whether these concerns are realistic given the results of empirical studies on the relationship between patents and biomedical research. Part IV also examines whether the district court’s holding in AMP is consistent with the policy goals behind intellectual property rights and the reality of the industry. Finally, this Note concludes that, in general, patents do not impede upon innovation. However, the broad issuance of composition claims, such as those held by Myriad in AMP, may block research in areas of study that the patent holder is not pursuing (such as therapeutics). This Note suggests that this issue could be resolved by narrowing the focus of the patent claim to the application of the gene composition, rather than the composition on its own.

Transforming Properties of 8p11-12 Amplified Genes in Human Breast Cancer

Amplification of the 8p11-12 region has been found in about 15% of human breast cancers and is associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon to identify candidate oncogenes in breast cancer. We identified 21 candidate genes and provided evidence that three genes, namely, LSM-1, TC-1, and BAG4, have transforming properties when overexpressed. In the present study, we systematically investigated the transforming properties of 13 newly identified 8p11-12 candidate oncogenes in vitro. WHSC1L1, DDHD2, and ERLIN2 were most potently transforming oncogenes based on the number of altered phenotypes expressed by the cells. WHSC1L1 contains a PWWP-domain that is a methyl-lysine recognition motif involved in histone code modification and epigenetic regulation of gene expression. Knockdown of WHSC1L1 in 8p11-12-amplified breast cancer cells resulted in profound loss of growth and survival of these cells. Further, we identified several WHSC1L1 target genes, one of which is iroquois homeobox 3 gene (IRX3), a member of the Iroquois homeobox transcription factor family.

Alteration of topoisomerase II-alpha gene in human breast cancer: Association with responsiveness to anthracycline-based chemotherapy.

518 Background: Approximately 35% of HER2-amplified breast cancers have coamplification of the topoisomerase II-alpha (TOP2A) gene which encodes an enzyme that is a major target of anthracyclines. This study determines whether TOP2A rather than HER2 gene alterations may be predictive for unique responsiveness to anthracyclines in some breast cancers. Methods: Approximately 5,000 breast cancers were analyzed for alterations in TOP2A and HER2. Using fluorescence in situ hybridization, a test set of tumor tissues from metastatic breast cancer patients treated in a study of chemotherapy with or without trastuzumab and subsequent, large validation set trials were evaluated for amplification or deletion of these genes. Association between these alterations and clinical outcomes was determined. Results: The test set demonstrated that cases containing HER2 amplification treated with doxorubicin, cyclophosphamide (AC) plus trastuzumab had a longer progression-free survival compared to those treated with AC alone (p = 0.03). However, patients treated with AC alone whose tumors contain HER2/TOP2A coamplification experienced a similar significant increase in survival compared to those with only HER2 amplification (p = 0.004). Conversely, in patients treated with paclitaxel, HER2/TOP2A coamplification was not associated with improved outcomes. These observations were confirmed in validation set cases, where HER2/TOP2A coamplification was again associated with longer survival when anthracycline-containing chemotherapy was used compared to cancers lacking TOP2A coamplification. Conclusions: Both the test set and validation set demonstrate that TOP2A coamplification, not HER2 amplification, is the clinically useful predictive marker of a large increment in response to anthracycline-based chemotherapy in a small number of breast cancer patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration IDDI AstraZeneca, Genentech, GlaxoSmithKline, Merck, Novartis, Roche, sanofi-aventis, Ventana Medical Sysems IDDI AstraZeneca, Genentech, Merck, Novartis, Roche, sanofi-aventis Genentech, GlaxoSmithKline, Roche, sanofi-aventis, Ventana Medical Systems

Mammary Tumor Development in Dogs Is Associated with BRCA1 and BRCA2

Breast cancer is a major contributor to overall morbidity and mortality in women. Several genes predisposing to breast cancer have been identified, but the majority of risk factors remain unknown. Even less is known about the inherited risk factors underlying canine mammary tumors (CMT). Clear breed predispositions exist, with 36% of English springer spaniels (ESS) in Sweden being affected. Here, we evaluate 10 human breast cancer genes (BRCA1, BRCA2, CHEK2, ERBB2, FGFR2, LSP1, MAP3K1, RCAS1, TOX3, and TP53) for association with CMTs. Sixty-three single-nucleotide polymorphisms (SNPs; four to nine SNPs per gene) were genotyped by iPLEX in female ESS dogs, 212 CMT cases and 143 controls. Two genes, BRCA1 and BRCA2, were significantly associated with CMT (Bonferroni corrected P = 0.005 and P = 0.0001, respectively). Borderline association was seen for FGFR2. Benign and malignant cases were also analyzed separately. Those findings supported the association to BRCA1 and BRCA2 but with a stronger association to BRCA1 in malignant cases. Both BRCA1 and BRCA2 showed odds ratios of approximately 4. In conclusion, this study indicates that BRCA1 and BRCA2 contribute to the risk of CMT in ESS, suggesting that dogs may serve as a good model for human breast cancer.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin αvβ3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Expression of melanocyte-related genes in human breast cancer and its implications

We report the expression of melanocyte-related genes (MRG) in freshly resected, histopathologically confirmed, human breast cancer specimens and describe experiments illuminating similar observations on a variety of breast cancer cell lines including MDA-MB-435. This finding has implications for research on breast cancer, for clinical investigation of cancer patients presenting with metastases from occult primary tumors and for understanding aberrant differentiation in cancer cells. For example, higher expression of six MRG correlated inversely with propensity for metastatic spread in clones isolated from the human breast cancer cell line MDA-MB-435. Comparisons of MRG expression in cells growing in vitro with those seen in tumors generated by the same lines in vivo showed that the levels of activity of these genes are influenced by the surrounding environment. Also, silencing of expression of the melanocyte-related transcription factor MITF, by transduction of the non-metastatic clone NM2C5 with a construct expressing a specific anti-MITF shRNA, resulted in decreased production of 5 of the melanocyte-related proteins including TYRP1, Pmel 17, MART 1(Melan-A) and TYRP2, but no increase in metastatic capability. Hence MRG expression reproducibly ear-marked, but did not cause, metastatic incompetence. We also report cytogenetic and other data that conflict with the recent suggestion that MDA-MB-435 is of melanocytic origin and are more consistent with its original designation as being of mammary lineage. We conclude that detection of MRG expression profiles in freshly excised breast cancers and in cultured breast cancer cells reflects the operationally important clinical phenomenon of inappropriate gene expression in malignant neoplasms. Concomitantly, we suggest that the evidence we have obtained (i) collectively supports the continued widespread use of the MDA-MB-435 cell line in breast cancer and metastasis research and (ii) advances knowledge of the diversity of aberrant differentiation programs in malignant cells, which is valuable for making accurate diagnoses and treatment decisions in clinical oncology.