Marybeth A. Pysz

Protein kinase C-mediated down-regulation of cyclin D1 involves activation of the translational repressor 4E-BP1 via a phosphoinositide 3-kinase/Akt-independent, protein phosphatase 2A-dependent mechanism in intestinal epithelial cells.

We reported previously that protein kinase Cα (PKCα), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKCα-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr45 and Ser64 on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKCα-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an ∼2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H2O2 and ceramide, two naturally occurring PKCα agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKCα-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/PKCα-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells.

Involvement of the ERK Signaling Cascade in Protein Kinase C-mediated Cell Cycle Arrest in Intestinal Epithelial Cells

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins (Frey, M. R., Clark, J. A., Leontieva, O., Uronis, J. M., Black, A. R., and Black, J. D. (2000) J. Cell Biol. 151, 763–777). In the current study, we present evidence to support a requisite role for PKC α in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC α activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC α activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC α activation, including cyclin D1 down-regulation, p21Waf1/Cip1 induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC α activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC α activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC α and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.

Protein Kinase C α Signaling Inhibits Cyclin D1 Translation in Intestinal Epithelial Cells

Cyclin D1 is a key regulator of cell proliferation, acting as a mitogen sensor and linking extracellular signaling to the cell cycle machinery. Strict control of cyclin D1 levels is critical for maintenance of tissue homeostasis. We have reported previously that protein kinase C α (PKCα), a negative regulator of cell growth in the intestinal epithelium, promotes rapid down-regulation of cyclin D1 (Frey, M. R., Clark, J. A., Leontieva, O., Uronis, J. M., Black, A. R., and Black, J. D. (2000) J. Cell Biol. 151, 763–778). The current study explores the mechanisms underlying PKCα-induced loss of cyclin D1 protein in non-transformed intestinal epithelial cells. Our findings exclude several mechanisms previously implicated in down-regulation of cyclin D1 during cell cycle exit/differentiation, including alterations in cyclin D1 mRNA expression and protein turnover. Instead, we identify PKCα as a novel repressor of cyclin D1 translation, acting at the level of cap-dependent initiation. Inhibition of cyclin D1 translation initiation is mediated by PKCα-induced hypophosphorylation/activation of the translational suppressor 4E-BP1, association of 4E-BP1 with the mRNA cap-binding protein eIF4E, and sequestration of cyclin D1 mRNA in 4E-BP1-associated complexes. Together, these post-transcriptional effects ensure rapid disappearance of the potent mitogenic molecule cyclin D1 during PKCα-induced cell cycle withdrawal in the intestinal epithelium.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells To Result In Sustained Tumor Regressions

Purpose: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. Experimental Design: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody–drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. Results: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. Conclusions: These findings demonstrate the potential of PF-06647263 (anti–EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development. Clin Cancer Res; 21(18); 4165–73. ©2015 AACR.

PKCα tumor suppression in the intestine is associated with transcriptional and translational inhibition of cyclin D1

Alterations in PKC isozyme expression and aberrant induction of cyclin D1 are early events in intestinal tumorigenesis. Previous studies have identified cyclin D1 as a major target in the antiproliferative effects of PKCalpha in non-transformed intestinal cells; however, a link between PKC signaling and cyclin D1 in colon cancer remained to be established. The current study further characterized PKC isozyme expression in intestinal neoplasms and explored the consequences of restoring PKCalpha or PKCdelta in a panel of colon carcinoma cell lines. Consistent with patterns of PKC expression in primary tumors, PKCalpha and delta levels were generally reduced in colon carcinoma cell lines, PKCbetaII was elevated and PKCepsilon showed variable expression, thus establishing the suitability of these models for analysis of PKC signaling. While colon cancer cells were insensitive to the effects of PKC agonists on cyclin D1 levels, restoration of PKCalpha downregulated cyclin D1 by two independent mechanisms. PKCalpha expression consistently (a) reduced steady-state levels of cyclin D1 by a novel transcriptional mechanism not previously seen in non-transformed cells, and (b) re-established the ability of PKC agonists to activate the translational repressor 4E-BP1 and inhibit cyclin D1 translation. In contrast, PKCdelta had modest and variable effects on cyclin D1 steady-state levels and failed to restore responsiveness to PKC agonists. Notably, PKCalpha expression blocked anchorage-independent growth in colon cancer cells via a mechanism partially dependent on cyclin D1 deficiency, while PKCdelta had only minor effects. Loss of PKCalpha and effects of its re-expression were independent of the status of the APC/beta-catenin signaling pathway or known genetic alterations, indicating that they are a general characteristic of colon tumors. Thus, PKCalpha is a potent negative regulator of cyclin D1 expression and anchorage-independent cell growth in colon tumor cells, findings that offer important perspectives on the frequent loss of this isozyme during intestinal carcinogenesis.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions

PTK7 is a tumor-initiating cell antigen, which can be targeted with an antibody-drug conjugate to confer sustained tumor regressions. Initiating an antitumor attack Cancer is notorious for relapsing after treatment, making it difficult to eradicate from a patient’s body. Such relapses are driven by tumor-initiating cells, a type of stem cells that give rise to tumors. Damelin et al. determined that a protein called PTK7 is frequently present on tumor-initiating cells and developed an antibody-drug conjugate for targeting it. The authors demonstrated the effectiveness of this therapy in mouse models of several tumor types and confirmed that it reduces tumor-initiating cells and outperforms standard chemotherapy. The antibody-drug conjugate also had some unexpected benefits, reducing tumor angiogenesis and promoting antitumor immunity, all of which may contribute to its effectiveness. Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non–small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient–derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline–based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Differential Regulation of Cyclin D1 Expression by Protein Kinase C α and ϵ Signaling in Intestinal Epithelial Cells

Background: Tight control of cyclin D1 expression is critical for intestinal homeostasis. Results: Whereas PKCα suppresses cyclin D1 expression, PKCϵ up-regulates cyclin D1 via an ERK and NF-κB/CRE-mediated transcriptional mechanism. Conclusion: Cyclin D1 levels in intestinal cells reflect a balance between PKCα and PKCϵ signaling. Significance: The opposing effects of PKCα and PKCϵ on cyclin D1 accumulation reflect their contrasting contributions to intestinal tumorigenesis. Cellular accumulation of cyclin D1, a key regulator of cell proliferation and tumorigenesis, is subject to tight control. Our previous studies have identified PKCα as a negative regulator of cyclin D1 in the intestinal epithelium. However, treatment of non-transformed IEC-18 ileal crypt cells with PKC agonists has a biphasic effect on cyclin D1 expression. Initial PKCα-mediated down-regulation is followed by recovery and subsequent accumulation of the cyclin to levels markedly higher than those seen in untreated cells. Using protein overexpression strategies, siRNA, and pharmacological inhibitors, we now demonstrate that the recovery and hyperinduction of cyclin D1 reflect the combined effects of (a) loss of negative signals from PKCα due to agonist-induced PKCα down-regulation and (b) positive effects of PKCϵ. PKCϵ-mediated up-regulation of cyclin D1 requires sustained ERK stimulation and transcriptional activation of the proximal cyclin D1 (CCDN1) promoter, without apparent involvement of changes in protein stability or translation. PKCϵ also up-regulates cyclin D1 expression in colon cancer cells, through mechanisms that parallel those in IEC-18 cells. Although induction of cyclin D1 by PKCϵ is dependent on non-canonical NF-κB activation, the NF-κB site in the proximal promoter is not required. Instead, cyclin D1 promoter activity is regulated by a novel interaction between NF-κB and factors that associate with the cyclic AMP-response element adjacent to the NF-κB site. The differential effects of PKCα and PKCϵ on cyclin D1 accumulation are likely to contribute to the opposing tumor-suppressive and tumor-promoting activities of these PKC family members in the intestinal epithelium.

Abstract 3093: Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC)

Expression of DLL3 was examined in additional tumor types, as it was found to be highly expressed in tumor-initiating cells (TIC) in small cell lung cancer (SCLC), where a DLL3-targeted antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T; SC16LD6.5; Saunders et al. 2015 Sci Transl Med 7:302ra136)) exerted clinically meaningful anti-tumor effects in a phase I trial (Spigel et al. 2016 Lancet Oncology; In Press). DLL3 expression was profiled by qRT-PCR, ELISA and immunohistochemistry in multiple tumor types. Patient-derived xenografts (PDX) from melanoma and ovarian small cell carcinoma were established and used for efficacy studies to determine the ability of Rova-T to impact tumor growth and TIC frequency. DLL3 expression was seen in metastatic melanoma (55%), low grade gliomas (90%), glioblastoma (70%), medullary thyroid cancer (65%), carcinoids (33%), dispersed neuroendocrine tumors in the pancreas (9%), bladder (57%) and prostate (24%), testicular cancer (90%), and lung adenocarcinomas with neuroendocrine features (80%). Unlike SCLC, where DLL3 does not predict clinical outcome on standard therapies, DLL3 expression negatively correlates with overall survival in melanoma and small cell bladder cancer. In mice bearing DLL3 positive melanoma PDX, treatment with a single dose of Rova-T resulted in effective and durable responses (>100 days), which correlated with a significant impact on TIC frequency. Similarly, in mice bearing DLL3-positive ovarian small cell PDX, a single dose of Rova-T resulted in effective and durable responses (>100 days). Our results show that DLL3 is expressed in many neuroendocrine tumors (lung, ovarian, prostate, bladder, etc), metastatic melanoma, medullary thyroid cancer, low-grade gliomas and glioblastoma. Given pre-clinical results showing efficacy of Rova-T in melanoma and ovarian small cell carcinoma, as well as encouraging clinical data with Rova-T in patients with recurrent/refractory SCLC, clinical evaluation of Rova-T in DLL3-positive melanoma, glioblastoma, medullary thyroid cancer and other high-grade neuroendocrine carcinomas is warranted. A “basket” trial enrolling patients with DLL3-positive solid tumors is now recruiting patients (NCT02709889). Citation Format: Laura R. Saunders, Samuel A. Williams, Sheila Bheddah, Kumiko Isse, Sarah Fong, Marybeth A. Pysz, Himisha Beltran, Loredana Puca, Verena Sailer, Juan M. Mosquera, Yu Yin, Jiaoti Huang, Andrew J. Armstrong, Jorge Garcia, Cristina Magi-Galluzzi, Vadim Koshkin, Petros Grivas, Farhad Kosari, John Cheville, Justin C. Moser, Thomas J. Flotte, Thorvardur Halfdanarson, Aaron Mansfield, Konstantinos N. Leventakos, Julian R. Molina, Douglas W. Ball, Barry D. Nelkin, Jill E. Shea, Courtney L. Scaife, Scott J. Dylla. Expression of DLL3 in metastatic melanoma, glioblastoma and high-grade extrapulmonary neuroendocrine carcinomas as potential indications for rovalpituzumab tesirine (Rova-T; SC16LD6.5), a delta-like protein 3 (DLL3)-targeted antibody drug conjugate (ADC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2017-3093

Abstract 293: Regulation of inhibitor of differentiation 1 (Id1) by PKC signaling in the intestinal epithelium

Increasing evidence supports a role for protein kinase C (PKC) signaling in regulation of intestinal epithelial self-renewal, and alterations in PKC isozyme expression/activity have been broadly linked to intestinal carcinogenesis. PKCα, which exhibits growth inhibitory and tumor suppressor properties in the intestine, is lost early during intestinal tumor development. To gain further insight into the tumor suppressive effects of PKCα signaling, we explored the consequences of restoration of PKCα expression in DLD-1 human colon cancer cells by microarray analysis. The most downregulated known gene in PKCα-expressing DLD-1 cells was inhibitor of differentiation 1 (Id1), a member of the Id family of dominant negative antagonists of bHLH transcription factors that has cell cycle stimulatory functions and has been associated with tumorigenesis in a number of systems. Suppression of Id1 protein by PKCα in colon cancer cells was confirmed by Western blot analysis in a panel of cell lines including DLD-1, HCT116, FET, FET/DNR, and GEO. Notably, activation of PKC/PKCα in non-transformed IEC-18 intestinal crypt cells also resulted in rapid and profound downregulation of Id1 mRNA and protein, pointing to a role for PKCα in Id1 regulation in the normal intestinal epithelium. PKC-induced suppression of Id1 was found to occur via an ERK/MAPK-dependent, PI3K-independent mechanism. Manipulation of Id1 levels in normal and transformed intestinal cells demonstrated its ability to affect two key cell cycle targets of PKCα, the potent mitogen cyclin D1 and the cdk inhibitor p21 Waf1/Cip1 . Knockdown of Id1 consistently decreased steady-state levels of cyclin D1, while overexpression of the factor resulted in reduced levels of p21 Waf1/Cip1 . Thus, Id1 appears to control the expression of important cell cycle regulators in intestinal cells. Immunohistochemical analysis of normal intestinal mucosa further demonstrated that PKCα activation, which occurs precisely at the point of growth arrest in the mid/upper crypt, coincides with marked downregulation of Id1 in vivo. Conversely, loss of PKCα in intestinal tumors is associated with Id1 overexpression. Ongoing studies are focused on delineating the link between the tumor suppressor functions of PKCα and its inhibitory effects on Id1 expression in intestinal cells. Supported by NIH grants DK54909, DK60632, CA16506, and CA113048. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 293.

Abstract 4937: Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development

Patient derived xenograft (PDX) models of pancreatic cancer provide an excellent platform for understanding human disease, characterizing tumor-initiating cells, and testing of novel therapeutics. Herein, we compared the natural pancreatic microenvironment via orthotopic implantation and the traditional approach of subcutaneous implantation for effects on PDX tumor growth, tumor morphology, cell population diversity, tumorigenic potential, metastatic potential, and response to therapeutics. Two approaches were used: (1) primary human cancer tissue fragments were implanted in orthotopic and subcutaneous environments; and, (2) PDX previously established by subcutaneous engraftment were reimplanted into either an orthotopic or a subcutaneous environment. We report that orthotopically-engrafted primary PDX more closely reflect the gross tumor morphology of the original patient than subcutaneously-derived tumors. In models derived from subcutaneous engraftment, variation of tumor morphology, tumor growth, cell population diversity, and tumorigenic potential between tumors grown in orthotopic and subcutaneous environments were dependent on PDX line; and, stromal infiltration and mucin production were more prominent in the orthotopic background for some PDX models. In addition, we observe increased metastatic potential in orthotopic tumor-bearing mice compared with their subcutaneous counterparts. Tumor metastasis occurred in distal filtering organs, including the lung, liver, lymph nodes, and peritoneal cavity, among others. This result is consistent with the observation of increased circulating tumor cells in the blood of orthotopic tumor bearing mice. Comparison of therapeutic efficacy with gemcitabine chemotherapy and antibody-drug conjugate (ADC) therapy in orthotopic and subcutaneous tumors showed therapeutic response was dependent on PDX line. Interestingly, for PDX lines that exhibited morphological differences between orthotopic and subcutaneous backgrounds, treatment was less effective in the orthotopic microenvironment suggesting the microenvironment alters the ability of therapeutics to impede tumor growth. Reduced therapeutic response was also found to be consistent with reduced ADC uptake and vascularity in orthotopic tumors. Taken together, the orthotopic PDX model serves as a more accurate representation of human pancreatic cancer by displaying stromal-rich morphology, exhibiting metastatic cell behavior, and recapitulating therapeutic response challenges observed in patients. Citation Format: Sarah L. Fong, Marybeth A. Pysz, Kristen D. McKnight, Nicole Taylor, Regina Nieu, Juliet Markeson, Hanna Rammoth, Archana Dilip, Kayla Fasano, Emily Janke, Erica Anderson, Holger Karsunky, Scott Dylla. Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2017-4937