Latest LOBULAR news from the 2016 AACR conference

JohnSmith

The annual AACR (American Association for Cancer Research) conference starts in a couple weeks started!
16-20 April 2016 - New Orleans, Louisiana, USA

Focused on cancer research, the theme will revolve around the red hot topic of Immuno-Oncology and Immunotherapy drugs, which are generating durable remissions in a variety of different cancer patients.
The Breast Cancer industry is represented at this meeting.
I reviewed this years Program and discovered 6 sessions dedicated to ILC research. This is encouraging, considering the meeting represents all ~300 forms of cancer. By comparison, last years 2015 SABCS had only 10 sessions focused on ILC.

Below are the 6 ILC topics, followed by more details.

1. WNT4 mediates endocrine response and resistance in invasive lobular carcinoma cell lines and patient tumor explants
2. Multiplatform molecular profiling of invasive lobular breast cancer
3. The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma
4. Systematic in vivo analysis of PI3K pathway aberrations in a mouse model of invasive lobular carcinoma
5. Rapid in vivo testing of tumor suppressors in ILC by CRISPR-Cas9 mediated somatic gene editing of the mammary gland
6. Synthetic lethal approaches targeting E-cadherin-deficient cancers

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Category: Molecular Pharmacology of Hormone-dependent Malignancies [Minisymposium]
1. Abstract 862: WNT4 mediates endocrine response and resistance in invasive lobular carcinoma cell lines and patient tumor explants
When: Sunday, Apr 17, 2016, 4:50 PM - 5:05 PM
Authors: Matthew J. Sikora1, Courtney L. Andersen1, Caroline M. Alexander2, Priscilla M. McAuliffe1, Steffi Oesterreich1.
1University of Pittsburgh, Pittsburgh, PA; 2University of Wisconsin, Madison, WI

Abstract Body: Invasive lobular carcinoma (ILC) is a breast cancer subtype affecting ~30,000 U.S. women annually. Over 90% of ILC are estrogen receptor (ER)-positive; however, endocrine therapy may have poorer efficacy in a subset of ILC patients versus invasive ductal carcinoma (IDC) patients. This prompted us to assess global ER activity in ILC cell lines MDA MB 134VI (MM134) and SUM44PE (44PE) to identify novel mediators of ER signaling. These analyses identified the Wnt ligand WNT4 as an ILC-specific ER target gene, with an ILC-specific ER binding site (ERBS) at the WNT4 locus. Considering the critical role of WNT4 in normal mammary gland expansion, we hypothesize that ILC cells utilize WNT4 signaling to drive endocrine response and resistance.
We assessed whether WNT4 is necessary for ILC cell growth using siRNA. WNT4 knockdown completely blocked estrogen-induced growth in ILC cells but not IDC cells. In parallel, the WNT4 ERBS was only occupied in ILC cells in response to estrogen, but progesterone-induced WNT4 in IDC was not associated with this ERBS. This suggests that, via the ILC-specific WNT4 ERBS, ILC cells drive estrogen-regulated proliferation by hijacking a developmental Wnt pathway. Wnt pathways typically activate β-catenin; however, we observed β-catenin dysfunction in ILC cells and that WNT4 cannot activate β-catenin. Thus, WNT4 signals in ILC cells via a novel non-canonical pathway.
Using long-term estrogen-deprived (LTED) variants of MM134 and 44PE (4 and 2 lines, respectively), we assessed WNT4 in ILC endocrine resistance. WNT4 is over-expressed, but uncoupled from ER, in all MM134-LTED. Conversely, WNT4 is reduced in 44PE-LTED but remains ER-regulated; ER occupies the WNT4 ERBS only in 44PE-LTED cells and not MM134-LTED. Using siRNA, MM134-LTED (high WNT4) are growth-inhibited by WNT4 knockdown, while 44PE-LTED (low WNT4) are insensitive. However, WNT4 knockdown sensitizes 44PE-LTED to endocrine therapy. Taken together, uncoupling and upregulating WNT4 or WNT4/ER cross-talk may represent convergent endocrine resistance mechanisms in ILC.
To assess the role of WNT4 in patient ILC, we examined WNT4 protein in archival breast tumors and observed that WNT4 is frequently expressed in ILC and IDC tumors. We also examined WNT4 regulation and endocrine response in patient tumor explants. We observed ER regulation of WNT4 directly in ILC tissues that correlated with sensitivity to fulvestrant but resistance to tamoxifen; this may mimic clinical endocrine resistance. Ongoing studies are assessing WNT4 as a biomarker and mediator of endocrine resistance in ILC.
Clinical observations suggest that ER regulates unique pathways in ILC. We identified WNT4 as a downstream effector of endocrine signaling in ILC, with critical roles in both estrogen-induced growth and endocrine resistance. WNT4 signaling may represent a novel target to modulate endocrine response specifically for ILC patients.

Category: Genomic Profiling of Cancers [Poster Session]
2. Abstract 123: Multiplatform molecular profiling of invasive lobular breast cancer
When: Sunday, Apr 17, 2016, 1:00 PM - 5:00 PM
Authors: Raquel A. Nunes1, David Arguello2, Zoran Gatalica2, Sandeep Reddy2, Sandra M. Swain1.
1Washington Hospital Ctr., Washington, DC; 2Caris Life Sciences, Phoenix, AZ

Abstract Body: Introduction: Invasive lobular breast cancer (ILC) is the second most common subtype of invasive breast cancer accounting for 10% of breast cancer diagnosis. ILC has particular histological and clinical characteristics and a distinct response to therapy. Characterizing the molecular alterations in ILC may lead to an improved understanding of its biology and provide new therapeutic options. The purpose of this study is to describe the molecular profile of ILC and compare it to the one of invasive ductal cancer (IDC).
Methods: Three-hundred and thirty nine pure ILC specimens profiled from January 2012 - November 2015 were evaluated (Caris Life Sciences, Phoenix, AZ). Multiplatform profiling consisted of gene sequencing (next generation sequencing [NGS]), gene amplification (CISH or FISH), and protein expression (immunohistochemistry [IHC]). Molecular characteristics of estrogen receptor (ER) positive and human epidermal growth receptor factor 2 (HER2) negative pure ILC (n= 236) and IDC (n=286) were compared.
Results: By IHC, ER expression was present in 87.7% (277/316), progesterone receptor in 59.6% (198/313), HER2 in 3.5% (11/313), androgen receptor in 87% (262/301), PD-L1 in 8.1% (12/148) and PTEN in 63.3% (198/313). Amplifications were detected in MYC (7.7%, 2/26), EGFR (8.3%, 2/24), ERBB2 (4.5%, 13/290) and TOP2A (1.3%, 3/236). Mutations were detected in AKT1 (4.7%, 9/191), ATM (3.7%, 7/190), BRCA1 (4.2%, 4/96), BRCA2 (9.5%, 9/95), ERBB2 (7.5%, 14/186), PIK3CA (54.5%, 103/189), PTEN (7.9%, 15/189), and TP53 (13.4%, 25/186). A comparison of ER-positive/HER2-negative invasive lobular and ductal carcinomas revealed significant differences in AR expression (89.7% vs 79.6%, p = 0.0022), and mutations in CDH1 (10.1% vs 0.0%, p = 0.0001), ERBB2 (8.2% vs 2.1%, p = 0.0079), and TP53 (10.3% vs 31.8%, 0.0001).
Conclusions: Multiplatform testing of this large series of ILC reveals recurrent alterations and a distinct molecular profile when compared to IDC. These support the definition of ILC as biologically distinct entity. High AR expression and high rates of dysregulation along the PIK3CA/AKT/mTOR pathway are consistent with recent reports in the literature.

Category: Mechanisms of Tumorigenesis in Animal Models of Cancer 1 [Poster Session]
3. Abstract 673: The role of MYPT1/2, ASPP2 and MYH9 in invasive lobular carcinoma
When: Sunday, Apr 17, 2016, 1:00 PM - 5:00 PM
Authors: Koen Schipper1, Julian de Ruiter1, Sjors Kas1, Eva Schut1, Marco Koudijs1, David Addams2, Jos Jonkers1.
1NKI-AVL, Amsterdam, Netherlands; 2Sanger institute, Cambridge, United Kingdom

Abstract Body: Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of this type of cancer is the loss of intercellular adhesion which is facilitated by inactivating mutations in E-cadherin. However the loss of E-cadherin alone is not enough to induce ILC. An in vivo Sleeping Beauty insertional mutagenesis screen was performed to identify genes that together with E-cadherin loss contribute to ILC development in mice. In around 85% of the tumors that were analyzed during this screen one of four hits were observed, namely MYPT1, MYPT2, ASPP2 and MYH9. Interestingly these four hits appear to be mutually exclusive indicating a shared mechanism of action.
To investigate how these genes might affect tumorigenesis we first looked at the location of the transposon insertions. This revealed that for MYPT1, MYPT2 and ASPP2 the transposons appeared to localize to a specific region in the gene indicating that these insertions could result in a truncation variant. In the case of MYH9 no clear localization of insertions was found which in combination with the recently published data indicates a loss of function. Northern blot analysis revealed the presence of truncation variants for MYPT1/2 and ASPP2 which for MYPT1 were confirmed to also result in truncated protein variants.
In order to identify the tumorigenic potential of truncated MYPT1 and ASPP2 we overexpressed them in spontaneously immortalized mammary epithelial cell lines HC11 and NMuMG. We are using these cell lines to analyse differences in cell proliferation, anchorage independent growth, sensitivity to apoptosis in vitro and tumor formation in vivo. To further analyse the hits in an E-cadherin negative setting we made use of primary mammary epithelial cells (MECs) isolated from genetically engineered mice with mammary gland specific E-cadherin loss. These MECs normally do not grow past several passages in vitro. However, these MECs when we overexpress truncated MYPT1 or ASPP2, or use shRNA mediated knockdown of MYH9 can be passaged and grown in vitro consistently.
Finally we are generating conditional mouse models with mammary gland specific E-cadherin loss in combination of expression of the truncated variants of MYPT1 or ASPP2, or loss of MYH9. We will to monitor whether these mice are prone to developing ILC.

Category: Mechanisms of Tumorigenesis in Animal Models of Cancer 2 [Poster Session]
4. Abstract 4169: Systematic in vivo analysis of PI3K pathway aberrations in a mouse model of invasive lobular carcinoma
When: Tuesday, Apr 19, 2016, 1:00 PM - 5:00 PM
Authors: Martine Van Miltenburg.
Netherlands Cancer Inst., Amsterdam, Netherlands

Abstract Body: Invasive lobular carcinoma (ILC) is the second most prevalent breast cancer subtype, which develops in the milk-producing glands (lobules) and metastasizes to distant organs. A key feature of ILC is loss of the CDH1 gene, encoding the E-cadherin cell-cell adhesion receptor. Though E-cadherin is thought to be a tumour suppressor, it is interesting to note that mammary-gland specific deletion of E-cadherin does not lead to tumour development indicating that other factors are necessary for the induction of ILC. Recent data from the cancer genome atlas reveals that besides E-cadherin loss, mutations in PIK3CA, and to a lesser extent PTEN, TBX3, and FOXA1 were found as ILC enriched features. These data implicate PI3K signaling as a driving force in ILC development. Using our in-house established GEMM-ESC strategy, we have developed innovative mouse models for ILC to study novel aspects of the functional interplay between E-cadherin and PI3K in ILC development. We have discovered an intriguing new interplay between these components in which E-cadherin loss suppresses growth of normal mammary epithelium, which appears to involve senescence, whereas it effectively accelerates tumor growth in a PIK3CAmutant background. Therapeutically, PIK3CAE545K and AKTE17K ILCs were sensitive to AZD8055, an mTOR inhibitor, and BKM120, a PI3K 110α inhibitor in vivo, indicating that PI3K signaling is required for their tumor maintenance. Currently, we are dissecting the sensitivity of ILCs displaying PIK3CAE545K or AKTE17K mutations, and PTEN loss, to a large panel of inhibitors, which will likely provide new clinical options.

Category: Mechanisms of Tumorigenesis in Mouse Models of Human Cancer [Minisymposium]
5. Abstract 2687: Rapid in vivo testing of tumor suppressors in ILC by CRISPR-Cas9 mediated somatic gene editing of the mammary gland
Monday, Apr 18, 2016, 4:05 PM - 4:20 PM
Stefano Annunziato, Sjors Kas, Micha Nethe, Hatice Yucel, Jessica del Bravo, Colin Pritchard, Rahmen Bin Ali, Bas van Gerwen, Bjorn Siteur, Anne Paulien Drenth, Eva Schut-Kregel, Sjoerd Klarenbeek, Ivo Huijbers, Martine van Miltenburg, Jos Jonkers.
NKI-AVL, Amsterdam, Netherlands

Abstract Body: Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype, accounting for 5% to 15% of breast tumors.The majority of ILCs are characterized by the complete loss of the cell adhesion protein E-cadherin encoded by the CDH1 gene. However, WAPcre;Cdh1F/F mice with mammary gland-specific E-cadherin loss do not develop ILC, unless coupled with the additional disruption of a tumor suppressor gene, like Pten or Trp53. Compound mutant mice develop lesions that closely resemble the human disease in terms of histology and invasivity. However, genome-wide sequencing projects and forward genetic screens identified a number of additional putative ILC-initiating loci. Hence, there is now an urgent need for validation and characterization of these candidate cancer genes. We sought to determine if it was possible to deploy CRISPR/Cas9 technology to somatically inactivate candidate tumor suppressor genes in mammary gland tissue of adult mice. For this purpose we performed in Cdh1F/F mice intraductal injections of high-titer lentiviral vectors carrying different combinations of Cre, Cas9 and sgRNAs targeting several candidate genes, including Pten, Tgfbr2, Myh9, Nf1 and Fbxw7. We found that Cas9-bearing vectors elicited strong immune responses against transduced cells, which resulted in small ILC lesions surrounded by immune cells. However, when Cas9 was expressed endogenously in the mammary tissue from a knock-in allele, intraductal injection of lentiviral vectors encoding the sgRNA moiety of the CRISPR system was sufficient to induce penetrant and multifocal ILC formation in WAPcre;Cdh1F/F;Cas9 female mice. Sequencing revealed specific mutations clustered in the CRISPR targeted gene, and positive selection for loss-of-function indels. Indeed, immunohistochemistry and immunofluorescence analysis confirmed that tumors were largely deficient for E-cadherin and the targeted gene. In sum, we have successfully established a new platform for in vivo CRISPR-Cas9 mediated somatic gene editing in the mouse mammary gland. This rapid and versatile platform can be used to identify novel tumor drivers (e.g.by employing forward genetic screens) and validate candidate cancer genes in ILC and other breast cancer types.

Category: Oncogenes and Tumor Suppressor Genes as Therapeutic Targets [Poster Session]
6. Abstract 3814: Synthetic lethal approaches targeting E-cadherin-deficient cancers
Tuesday, Apr 19, 2016, 1:00 PM - 5:00 PM
Augustine Chen1, Bryony J. Telford1, Andrew Single1, Henry Beetham1, Kaylene J. Simpson2, Parry Guilford1.
1University of Otago, Dunedin, New Zealand; 2Peter MacCallum Cancer Centre, Melbourne, Australia

Abstract Body: E-cadherin is a cellular adhesion protein that is frequently mutated in lobular breast cancer and diffuse gastric cancer. The E-cadherin protein which is encoded by the CDH1 gene has key roles in establishing and maintaining cell polarity and differentiation, the organization of cell migration and architecture and the mediation of signaling through various proliferation and survival pathways. E-cadherin also has a tumor suppressor role and its loss in cancer cells would preclude drug targeting by conventional therapy. To circumvent this, we have taken a synthetic lethal approach to exploit any vulnerability created by the loss of E-cadherin. In the therapeutic setting, synthetic lethality refers to a combination of a mutated gene and a drug targeted at a second gene or protein causing cell death (specifically in cancer cells). To identify the vulnerabilities created by E-cadherin loss, we performed a genome-wide siRNA knockdown screen in isogenic MCF10A cell lines with and without E-cadherin expression. From the functional screen, we identified broad classes of G-protein-coupled receptor (GPCR) signaling proteins and families of cytoskeletal proteins which were highly enriched among the synthetic lethal candidates. Indeed, we identified drug classes with linkages to several of the GPCR and cytoskeletal targets that showed evidence of E-cadherin synthetic lethality when we performed a 4,057 known drug screen. These included certain PI3K inhibitors (PI-103), anti-glucocorticoid (mifepristone), tyrosine kinase inhibitor (saracatinib) and multiple histone deacetylase inhibitors (vorinostat and entinostat). Interestingly, the combination of saracatinib and mifepristone gave a synergistic effect (combination index < 1.0) in targeting E-cadherin-deficient MCF10A cells. These results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both of sporadic and familial lobular breast cancer and diffuse gastric cancer.

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THANKS JohnSmith

JohnSmith

Today starts the American Association for Cancer Research (AACR) annual meeting (April 16-20).
During the next 5 days, new research and results from Oncology clinical trials will be presented, including immunotherapy and targeted therapy for breast cancer.
If you're on twitter, use ‪#‎AACR16‬ to follow the data dump.

Also, the 17th annual meeting of the American Society of Breast Surgeons (ASBrS) is ongoing as well (April 13-17). You can follow that conference using #ASBrS.

Sunnyone22

As always, your research is appreciated more than you'll ever know.

Thanks, John Smith.

Annette_U

Thanks John - I will show these to my molecular biologist Hubby so he can help translate them into simple terms I understand.

fizzdon52

Yes I wish I could decipher all that, don't have a hope in hell!

katykids

Agree! I couldn't really understand it either! Despite a year of library science degree. Perhaps that's why I never finished my Masters!

Annette_U

Fizz, Means We're ALIVE, still here to read all this gobbledygook!!!!!!!

maryland

Thanks for the info John, much of it does sound like gobblyguck but I can pull some sense out of it and it's nice to know they are separating us from IDC. Sounds as tho the future may bring us our own ILC specific tx's. Also helps explain the familiar side of it, I am not BRCA positive yet my grandmother and my mom's 2 sisters and 2 of my cousins all had BC, altho, I don't know which type.

Iwrite

Thank you John! This is interesting stuff.

What I pulled out of it was that ILC is marked by the decline or absence of E-Cadherin which is a cellular adhesion protein. The loss of E-cadherin suppresses growth of normal mammary epithelium which actively accelerates the PIK3CA mutation and tumor growth. They are looking at ways to boost E-caderin or somehow slow or stop the PIK3CA mutation growth in tests. Evidently the loss of E-cadherin is common to both ILC and gastric cancers.

Appreciate the download!