Phillip M. Pifer

Suppression of the Epithelial–Mesenchymal Transition by Grainyhead-like-2

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell-like compartment of tumors, including TGF-β dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-β receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.

Point Mutations in Aβ Result in the Formation of Distinct Polymorphic Aggregates in the Presence of Lipid Bilayers

A hallmark of Alzheimers disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimers disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.

The effect of set point ratio and surface Young’s modulus on maximum tapping forces in fluid tapping mode atomic force microscopy

There is great interest in using proximal probe techniques to simultaneously image and measure physical properties of surfaces with nanoscale spatial resolution. In this regard, there have been recent innovations in generating time-resolved force interaction between the tip and surface during regular operation of tapping mode atomic force microscopy (TMAFM). These tip/sample forces can be used to measure physical material properties of surface in an analogous fashion to the well-established static force curve experiment. Since its inception, it has been recognized that operation of TMAFM in fluids differs significantly from that in air, with one of the major differences manifested in the quality factor ( Q ) of the cantilever. In air, Q is normally on the order of 200–400, whereas in fluids, it is of the order of approximately 1–5. In this study, we explore the impact of imaging parameters, i.e., set point ratio and free cantilever oscillation amplitude, on time varying tip-sample force interactions in fluid TMAFM via simulation and experiment. The numerical AFM model contains a feedback loop, allowing for the simulation of the entire scanning process. In this way, we explore the impact of varying the Young’s modulus of the surface on the maximum tapping force.

Grainyhead-like 2 reverses the metabolic changes induced by the oncogenic epithelial-mesenchymal transition: effects on anoikis

Resistance to anoikis is a prerequisite for tumor metastasis. The epithelial-to-mesenchymal transition (EMT) allows tumor cells to evade anoikis. The wound-healing regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses/reverses EMT, accompanied by suppression of the cancer stem cell (CSC) phenotype and by resensitization to anoikis. Here, the effects of GRHL2 upon intracellular metabolism in the context of reversion of the EMT/CSC phenotype, with a view toward understanding how these effects promote anoikis sensitivity, were investigated. EMT enhanced mitochondrial oxidative metabolism. Although this was accompanied by higher accumulation of superoxide, the overall level of reactive oxygen species (ROS) declined, due to decreased hydrogen peroxide. Glutamate dehydrogenase 1 (GLUD1) expression increased in EMT, and this increase, via the product α-ketoglutarate (α-KG), was important for suppressing hydrogen peroxide and protecting against anoikis. GRHL2 suppressed GLUD1 gene expression, decreased α-KG, increased ROS, and sensitized cells to anoikis. Implications: These results demonstrate a mechanistic role for GRHL2 in promoting anoikis through metabolic alterations. Mol Cancer Res; 14(6); 528–38. ©2016 AACR.

Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial–mesenchymal transition

GRHL2 suppresses EMT to give a default epithelial phenotype. GRHL2 inhibits this process through the histone acetyltransferase coactivator p300, repressing the partial EMT and preventing induction of MMPs. The results demonstrate novel roles for p300 and GRHL2 in promoting or suppressing EMT in morphogenesis and tumor progression.

Comparative Analysis of the Views of Oncologic Subspecialists and Palliative/Supportive Care Physicians Regarding Advanced Care Planning and End-of-Life Care

Background: Early palliative/supportive care (PSC) consultation and advance care planning (ACP) improve outcomes for patients with incurable cancer. However, PSC is underutilized in the United States. Objective: To examine philosophical differences among PSC, radiation oncology (RO), and medical oncology (MO) physicians in order to understand barriers to early PSC referral. Design: An electronic survey collected views of a nationwide cohort of health-care professionals regarding ACP and end-of-life care. Setting/Participants/Measurements: A subgroup analysis compared the responses from all 51 PSC, 178 RO, and 81 MO physician participants (12% response rate), using Pearson χ2 and Mann-Whitney U tests for categorical and ordinal data, respectively. Results: More statistically significant differences were observed between RO-PSC (12 questions) and MO-PSC (12 questions) than RO-MO (4 questions). Both RO and MO were more likely than PSC physicians to believe doctors adequately care for emotional (P < .001) and physical (P < .001) needs of patients with an incurable illness. Both RO and MO were also less likely to believe that PSC physicians were helpful at addressing these needs (P = .002 and <.001, respectively) or that patients’ awareness of their life expectancy leads to better medical (P = .007 and .002, respectively) and personal (P = .001 for each) decisions. Palliative/supportive care physicians felt that doctors are generally less successful at explaining/clarifying advanced life-sustaining treatments than RO (P < .001) or MO (P = .004). MO favored later initiation of ACP than either RO (P = .006) or PSC physicians (P = .004). Conclusions: Differences in perception of appropriate end-of-life care exist between oncologists and PSC physicians, suggesting a need for improved education and communication between these groups.

The Lack of Consensus of International Contouring Guidelines for the Dorsal Border of the Chest Wall Clinical Target Volume: What is the Impact on Organs at Risk and Relationships to Patterns of Recurrence in the Modern-Era?

Purpose Variation exists in cooperative group recommendations for the dorsal border for the chest wall clinical target volume (CTV). We aimed to quantify the impact of this variation on doses to critical organs and examine patterns of chest wall recurrence relative to the pectoralis muscle. Methods and Materials We retrospectively assessed patterns of chest wall recurrence quantified to the recommended CTV borders for women treated between 2005 and 2017. We compared treatment plans for 5 women who were treated with left postmastectomy radiation therapy, with the chest wall contoured using varying dorsal borders for CTV: (1) Anterior pleural surface (Radiation Therapy Oncology Group), (2) anterior surface of pectoralis major (European Society for Radiotherapy and Oncology), and (3) anterior rib surface (institutional practice). Treatment plans were generated for 50 Gy in 25 fractions. Doses to organs-at-risk were compared using paired-sample t tests. Results Institutional patterns of chest wall recurrence were 64.7% skin and subcutaneous tissue, 23.5% both anterior to and between the pectoralis muscles, and 11.8% isolated to the tissue between the pectoralis major and minor. No chest wall recurrences were noted deep to pectoralis minor. When comparing the plans generated per the Radiation Therapy Oncology Group versus European Society for Radiotherapy and Oncology contouring guidelines, the mean lung V20Gy, heart mean dose, and left anterior descending artery mean dose were 33.5% versus 29.4% (P < .01), 5.2 Gy versus 3.2Gy (P = .02), and 27.3Gy versus 17.8Gy (P = .04), respectively. Conclusions The recommended variations in the dorsal chest wall CTV border have significant impact on doses to the heart and lungs. Although our study was limited by small numbers, our institutional patterns of recurrence would support a more anterior dorsal border for the chest wall CTV consistent with older literature.

Exceptional Eight-year Response to Stereotactic Radiosurgery Monotherapy for Multiple Brain Metastases

Breast cancer represents the second leading cause of brain metastases in women. Once diagnosed, brain metastases have been associated with a rapidly progressive and universally poor prognosis. Breast cancer patients, particularly those with advantageous disease characteristics, may achieve extended survival. This extended life expectancy highlights the importance of effective intracranial treatments that minimize treatment-related late toxicity. Whole brain radiation therapy (WBRT) remains a standard of care palliative option; however, concerns remain regarding the late neurocognitive effects. Stereotactic radiosurgery (SRS) provides dose-escalated radiation therapy over a shortened course, maintaining equivalent survival and minimizing normal brain tissue exposure. Herein, we present a breast cancer patient who demonstrated an exceptional response and remained functionally independent following 12 SRS courses targeting 14 unique brain metastases over eight years. The case illustrates the efficacy of SRS alone, as well as the comparable utility of multiple SRS treatment techniques (Gamma Knife (AB Elekta, Stockholm, Sweden), CyberKnife (Accuray, Sunnyvale, California), and TrueBeam (Varian Medical Systems, Palo Alto, California)).

Abstract 3436: Suppression of the epithelial-mesenchymal transition (EMT) by a wound-healing gene, Grainyhead-like-2

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of a Grainyhead gene (GRHL2) in oncogenic EMT. Grainyhead was specifically down-regulated in the claudin-low subclass of mammary tumors and in the basal-B subclass of breast cancer cell lines. Functionally, GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by its suppression of ZEB1 expression, by direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription, and up-regulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. The expression of GRHL2 in the breast cancer cell line MDA-MB-231 triggered a mesenchymal-to-epithelial transition and sensitized the cells to anoikis. These results indicate that GRHL2 is a suppressor of the oncogenic EMT, and provide a novel molecular focus for the wound-healing/oncogenic EMT relationship. 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 3436. doi:1538-7445.AM2012-3436

The Effect of Mutant Aβ Peptide Aggregation on the Stability of Model Lipid Bilayers

A hallmark of Alzheimers disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are five mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (the A21G Flemish mutation, E22K Italian mutation, E22G Arctic mutation, E22Q Dutch mutation and the D23N Iowa mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimers disease pathology with plaques and tangles. We hypothesizethat these point mutations alter the Aβ aggregation pathway and its interaction with cellular lipid membranes, resulting in altered disease progression and phenotypes. Brain lipid extract was used to form bilayers that are physiologically relevant models of neuronal cell surface. Intact lipid bilayers are exposed to different mutant forms of Aβ, and Atomic Force Microscopy was used to follow the aggregation of Aβ and membrane integrity over a 24 hour period. The goal of this study was to determine how point mutations in Aβ alter electrostatic interactions between the Aβ and the lipid surface. These interactions may affect aggregation, morphological characteristics, and bilayer disruption of Aβ on the model lipid membranes which may play a role in Aβ-related toxicity.