Erik S Knudsen
- Professor, Medicine
- Associate Director, Basic Sciences - Arizona Cancer Center
- Postdoctoral Fellow
- Cancer CenterUniversity of California San Diego, Spring 1998
- Department of BiologyUniversity of California San Diego, Spring 1997
No activities entered.
Introduction to ResearchMCB 795A (Fall 2016)
ResearchPCOL 900 (Fall 2016)
Research ConferencePCOL 695A (Fall 2016)
- Adams, T. A., Adams, T. A., Vail, P. J., Vail, P. J., Ruiz, A., Ruiz, A., Mollaee, M., Mollaee, M., McCue, P. A., McCue, P. A., Knudsen, E. S., Knudsen, E. S., Witkiewicz, A. K., & Witkiewicz, A. K. (2017). Composite analysis of immunological and metabolic markers defines novel subtypes of triple negative breast cancer. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc.More infoCancer biology is influenced by the tumor microenvironment, which impacts disease prognosis and therapeutic interventions. The inter-relationship of tumor-infiltrating lymphocytes, immune response regulators, and a glycolytic tumor environment was evaluated in a cohort of 183 largely consecutive patients with triple negative breast cancer diagnosis. High levels of tumor-infiltrating lymphocytes were associated with improved survival of triple negative breast cancer cases. However, elevated levels of PD-L1, CD163, and FOXP3 were individually associated with significantly decreased overall survival. These three determinants were significantly correlated, and could serve to differentiate the prognostic significance of tumor-infiltrating lymphocytes. Interestingly, a glycolytic tumor environment, as determined by the expression of MCT4 in the tumor stroma, was associated with the immune evasive environment and poor prognosis. Clustering of all markers defined four distinct triple negative breast cancer subtypes that harbored prognostic significance in multivariate analysis. Immune and metabolic markers stratified triple negative breast cancer into subtypes that have prognostic significance and implications for therapies targeting immune checkpoints and tumor metabolism.Modern Pathology advance online publication, 6 October 2017; doi:10.1038/modpathol.2017.126.
- Jansen, V. M., Bhola, N. E., Bauer, J. A., Formisano, L., Lee, K. M., Hutchinson, K. E., Witkiewicz, A. K., Moore, P. D., Estrada, M. V., Sánchez, V., Ericsson, P. G., Sanders, M. E., Pohlmann, P. R., Pishvaian, M. J., Riddle, D. A., Dugger, T. C., Wei, W., Knudsen, E. S., & Arteaga, C. L. (2017). Kinome-Wide RNA Interference Screen Reveals a Role for PDK1 in Acquired Resistance to CDK4/6 Inhibition in ER-Positive Breast Cancer. Cancer research, 77(9), 2488-2499.More infoAcquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR.
- Knudsen, E. S., & Witkiewicz, A. K. (2017). The Strange Case of CDK4/6 Inhibitors: Mechanisms, Resistance, and Combination Strategies. Trends in cancer, 3(1), 39-55.More infoCDK4/6 inhibitors have emerged as a powerful class of agents with clinical activity in a number of malignancies. Targeting the cell cycle represents a core attack on a defining feature of cancer. However, the mechanisms through which selective CDK4/6 targeted agents act has few parallels in the current pharmaceutical armamentarium against cancer. Notably, CDK4/6 inhibitors act downstream of most mitogenic signaling cascades, which have implications both related to clinical efficacy and resistance. Core knowledge of cell cycle processes has provided insights into mechanisms of intrinsic resistance to CDK4/6 inhibitors; however, the basis of acquired resistance versus durable response is only beginning to emerge. This review focuses on the mechanism of action and biomarkers to direct the precision use of CDK4/6 inhibitors and rationally-developed combination therapies.
- Knudsen, E. S., & Witkiewicz, A. K. (2017). The transcriptome of CDK4/6 inhibition. Aging, 9(8), 1859-1860.
- Knudsen, E. S., Balaji, U., Mannakee, B., Vail, P., Eslinger, C., Moxom, C., Mansour, J., & Witkiewicz, A. K. (2017). Pancreatic cancer cell lines as patient-derived avatars: genetic characterisation and functional utility. Gut.More infoPancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease with the worst survival rate of common solid tumours. Preclinical models that accurately reflect the genetic and biological diversity of PDAC will be important for delineating features of tumour biology and therapeutic vulnerabilities.
- Knudsen, E. S., Hutcheson, J., Vail, P., & Witkiewicz, A. K. (2017). Biological specificity of CDK4/6 inhibitors: dose response relationship, in vivo signaling, and composite response signature. Oncotarget, 8(27), 43678-43691.More infoRecently developed potent and selective CDK4/6 inhibitors fall into two classes based on structure and toxicity profiles in clinical studies. One class, exemplified by palbociclib and ribociclib, exhibits neutropenia as a dose-limiting toxicity and requires discontinuous dosing. In contrast, the structurally distinct CDK4/6 inhibitor abemaciclib is dosed continuously, and has diarrhea and fatigue as dose-limiting toxicities. In preclinical models, palbociclib has been extensively studied and induces cell cycle inhibition in an RB-dependent manner. Thus far, abemaciclib has been less extensively evaluated. We found that abemaciclib cell cycle inhibitory activity is RB-dependent at clinically achievable concentrations. Abemaciclib elicited potent suppression of RB/E2F regulated genes associated with prognosis in ER-positive breast cancer. However, unlike palbociclib, at 250nM-1 µM doses abemaciclib induced cell death in RB-deficient cell lines. This response was associated with a rapidly-induced multi-vacuolar phenotype indicative of lysosomal membrane permeabilization that could be ameliorated with chloroquine. This event was not a reflection of inhibition of other CDK family members, but could be recapitulated with CBX4945 that inhibits casein and DYRK/HIPK kinases. To determine if these "off-target" features of abemaciclib were observed in vivo, mice harboring matched RB-positive and negative xenografts were treated with palbociclib and abemaciclib. In vivo, all of the apparent activity of abemaciclib was RB-dependent and strongly elicited suppression of cell cycle regulatory genes in a fashion markedly similar to palbociclib. Using gene expression data from cell lines and tumors treated with abemaciclib and palbociclib a composite signature of response to CDK4/6 inhibition was developed that included many genes that are individually required for tumor cell proliferation or viability. These data indicate that while abemaciclib and palbociclib can exert distinct biological and molecular effects, there are common gene expression features that could be broadly utilized in measuring the response to CDK4/6 inhibition.
- Knudsen, E. S., Vail, P., Balaji, U., Ngo, H., Botros, I. W., Makarov, V., Riaz, N., Balachandran, V., Leach, S., Thompson, D. M., Chan, T. A., & Witkiewicz, A. K. (2017). Stratification of Pancreatic Ductal Adenocarcinoma: Combinatorial Genetic, Stromal, and Immunologic Markers. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(15), 4429-4440.More infoPurpose: Pancreatic ductal adenocarcinoma (PDAC) is associated with an immunosuppressive milieu that supports immune system evasion and disease progression. Here, we interrogated genetic, stromal, and immunologic features of PDAC to delineate impact on prognosis and means to more effectively employ immunotherapy.Experimental Design: A cohort of 109 PDAC cases annotated for overall survival was utilized as a primary discovery cohort. Gene expression analysis defined immunologic subtypes of PDAC that were confirmed in the Cancer Genome Atlas dataset. Stromal and metabolic characteristics of PDAC cases were evaluated by histologic analysis and immunostaining. Enumeration of lymphocytes, as well as staining for CD8, FOXP3, CD68, CD163, PDL1, and CTLA4 characterized immune infiltrate. Neoantigens were determined by analysis of whole-exome sequencing data. Random-forest clustering was employed to define multimarker subtypes, with univariate and multivariate analyses interrogating prognostic significance.Results: PDAC cases exhibited distinct stromal phenotypes that were associated with prognosis, glycolytic and hypoxic biomarkers, and immune infiltrate composition. Immune infiltrate was diverse among PDAC cases and enrichment for M2 macrophages and select immune checkpoints regulators were specifically associated with survival. Composite analysis with neoantigen burden, immunologic, and stromal features defined novel subtypes of PDAC that could have bearing on sensitivity to immunologic therapy approaches. In addition, a subtype with low levels of neoantigens and minimal lymphocyte infiltrate was associated with improved overall survival.Conclusions: The mutational burden of PDAC is associated with distinct immunosuppressive mechanisms that are conditioned by the tumor stromal environment. The defined subtypes have significance for utilizing immunotherapy in the treatment of PDAC. Clin Cancer Res; 23(15); 4429-40. ©2017 AACR.
- Franco, J., Balaji, U., Freinkman, E., Witkiewicz, A. K., & Knudsen, E. S. (2016). Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities. Cell reports, 14(5), 979-90.More infoDue to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models, CDK4/6 inhibition had a variable effect on cell cycle but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was associated with an increase in mTORC1 activity. MTOR and MEK inhibitors potently cooperate with CDK4/6 inhibition in eliciting cell-cycle exit. However, MTOR inhibition fully suppressed metabolism and yielded apoptosis and suppression of tumor growth in xenograft models. The metabolic state mediated by CDK4/6 inhibition increases mitochondrial number and reactive oxygen species (ROS). Concordantly, the suppression of ROS scavenging or BCL2 antagonists cooperated with CDK4/6 inhibition. Together, these data define the impact of therapeutics on PDA metabolism and provide strategies for converting cytostatic response to tumor cell killing.
- Hutcheson, J., Balaji, U., Porembka, M. R., Wachsmann, M. B., McCue, P. A., Knudsen, E. S., & Witkiewicz, A. K. (2016). Immunologic and Metabolic Features of Pancreatic Ductal Adenocarcinoma Define Prognostic Subtypes of Disease. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(14), 3606-17.More infoPancreatic ductal adenocarcinoma (PDA) is associated with an immunosuppressive microenvironment that supports the growth of the malignancy as well as immune system evasion. Here we examine markers of immunosuppression in PDA within the context of the glycolytic tumor microenvironment, their interrelationship with tumor biology and association with overall survival.
- Knudsen, E. S., & Witkiewicz, A. K. (2016). Defining the transcriptional and biological response to CDK4/6 inhibition in relation to ER+/HER2- breast cancer. Oncotarget, 7(43), 69111-69123.More infoER positive (ER+) and HER2 negative (HER2-) breast cancers are routinely treated based on estrogen dependence. CDK4/6 inhibitors in combination with endocrine therapy have significantly improved the progression-free survival of patients with ER+/HER2- metastatic breast cancer. Gene expression profiling in ER+/HER2- models was used to define the basis for the efficacy of CDK4/6 inhibitors and develop a gene expression signature of CDK4/6 inhibition. CDK4/6 inhibition robustly suppressed cell cycle progression of ER+/HER2- models and complements the activity of limiting estrogen. Chronic treatment with CDK4/6 inhibitors results in the consistent suppression of genes involved in cell cycle, while eliciting the induction of a comparable number of genes involved in multiple processes. The CDK4/6 inhibitor treatment shifted ER+/HER2- models from a high risk (luminal B) to a low risk (luminal A) molecular-phenotype using established gene expression panels. Consonantly, genes repressed by CDK4/6 inhibition are strongly associated with clinical prognosis in ER+/HER2- cases. This gene repression program was conserved in an aggressive triple negative breast cancer xenograft, indicating that this is a common feature of CDK4/6 inhibition. Interestingly, the genes upregulated as a consequence of CDK4/6 inhibition were more variable, but associated with improved outcome in ER+/HER2- clinical cases, indicating dual and heretofore unknown consequence of CDK4/6 inhibition. Interestingly, CDK4/6 inhibition was also associated with the induction of a collection of genes associated with cell growth; but unlike suppression of cell cycle genes this signaling was antagonized by endocrine therapy. Consistent with the stimulation of a mitogenic pathway, cell size and metabolism were induced with CDK4/6 inhibition but ameliorated with endocrine therapy. Together, the data herein support the basis for profound interaction between CDK4/6 inhibitors and endocrine therapy by cooperating for the suppression of cell cycle progression and limiting compensatory pro-growth processes that could contribute to therapeutic failure.
- Knudsen, E. S., Balaji, U., Freinkman, E., McCue, P., & Witkiewicz, A. K. (2016). Unique metabolic features of pancreatic cancer stroma: relevance to the tumor compartment, prognosis, and invasive potential. Oncotarget.More infoPancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. The aggressiveness and therapeutic recalcitrance of this malignancy has been attributed to multiple factors including the influence of an active desmoplastic stroma. How the stromal microenvironment of PDAC contributes to the fatal nature of this disease is not well defined. In the analysis of clinical specimens, we observed diverse expression of the hypoxic marker carbonic anhydrase IX and the lactate transporter MCT4 in the stromal compartment. These stromal features were associated with the epithelial to mesenchymal phenotype in PDAC tumor cells, and with shorter patient survival. Cultured cancer-associated fibroblasts (CAFs) derived from primary PDAC exhibited a high basal level of hypoxia inducible factor 1a (HIF1α) that was both required and sufficient to modulate the expression of MCT4. This event was associated with increased transcription and protein synthesis of HIF1α in CAFs relative to PDAC cell lines, while surprisingly the protein turnover rate was equivalent. CAFs utilized glucose predominantly for glycolytic intermediates, whereas glutamine was the preferred metabolite for the TCA cycle. Unlike PDAC cell lines, CAFs were resistant to glucose withdrawal but sensitive to glutamine depletion. Consistent with the lack of reliance on glucose, CAFs could survive the acute depletion of MCT4. In co-culture and xenograft studies CAFs stimulated the invasive potential and metastatic spread of PDAC cell lines through a mechanism dependent on HIF1α and MCT4. Together, these data indicate that stromal metabolic features influence PDAC tumor cells to promote invasiveness and metastatic potential and associate with poor outcome in patients with PDAC.
- Knudsen, E. S., O'Reilly, E. M., Brody, J. R., & Witkiewicz, A. K. (2016). Genetic Diversity of Pancreatic Ductal Adenocarcinoma and Opportunities for Precision Medicine. Gastroenterology, 150(1), 48-63.More infoPatients with pancreatic ductal adenocarcinoma (PDA) have a poor prognosis despite new treatments; approximately 7% survive for 5 years. Although there have been advances in systemic, primarily cytotoxic, therapies, it has been a challenge to treat patients with PDA using targeted therapies. Sequence analyses have provided a wealth of information about the genetic features of PDA and have identified potential therapeutic targets. Preclinical and early-phase clinical studies have found specific pathways could be rationally targeted; it might also be possible to take advantage of the genetic diversity of PDAs to develop therapeutic agents. The genetic diversity and instability of PDA cells have long been thought of as obstacles to treatment, but are now considered exploitable features. We review the latest findings in pancreatic cancer genetics and the promise of targeted approaches in PDA therapy.
- Witkiewicz, A. K., Balaji, U., Eslinger, C., McMillan, E., Conway, W., Posner, B., Mills, G. B., O'Reilly, E. M., & Knudsen, E. S. (2016). Integrated Patient-Derived Models Delineate Individualized Therapeutic Vulnerabilities of Pancreatic Cancer. Cell reports, 16(7), 2017-31.More infoPancreatic ductal adenocarcinoma (PDAC) harbors the worst prognosis of any common solid tumor, and multiple failed clinical trials indicate therapeutic recalcitrance. Here, we use exome sequencing of patient tumors and find multiple conserved genetic alterations. However, the majority of tumors exhibit no clearly defined therapeutic target. High-throughput drug screens using patient-derived cell lines found rare examples of sensitivity to monotherapy, with most models requiring combination therapy. Using PDX models, we confirmed the effectiveness and selectivity of the identified treatment responses. Out of more than 500 single and combination drug regimens tested, no single treatment was effective for the majority of PDAC tumors, and each case had unique sensitivity profiles that could not be predicted using genetic analyses. These data indicate a shortcoming of reliance on genetic analysis to predict efficacy of currently available agents against PDAC and suggest that sensitivity profiling of patient-derived models could inform personalized therapy design for PDAC.
- Asghar, U., Witkiewicz, A. K., Turner, N. C., & Knudsen, E. S. (2015). The history and future of targeting cyclin-dependent kinases in cancer therapy. Nature reviews. Drug discovery, 14(2), 130-46.More infoCancer represents a pathological manifestation of uncontrolled cell division; therefore, it has long been anticipated that our understanding of the basic principles of cell cycle control would result in effective cancer therapies. In particular, cyclin-dependent kinases (CDKs) that promote transition through the cell cycle were expected to be key therapeutic targets because many tumorigenic events ultimately drive proliferation by impinging on CDK4 or CDK6 complexes in the G1 phase of the cell cycle. Moreover, perturbations in chromosomal stability and aspects of S phase and G2/M control mediated by CDK2 and CDK1 are pivotal tumorigenic events. Translating this knowledge into successful clinical development of CDK inhibitors has historically been challenging, and numerous CDK inhibitors have demonstrated disappointing results in clinical trials. Here, we review the biology of CDKs, the rationale for therapeutically targeting discrete kinase complexes and historical clinical results of CDK inhibitors. We also discuss how CDK inhibitors with high selectivity (particularly for both CDK4 and CDK6), in combination with patient stratification, have resulted in more substantial clinical activity.
- Casimiro, M. C., Di Sante, G., Crosariol, M., Loro, E., Dampier, W., Ertel, A., Yu, Z., Saria, E. A., Papanikolaou, A., Li, Z., Wang, C., Addya, S., Lisanti, M. P., Fortina, P., Cardiff, R. D., Tozeren, A., Knudsen, E. S., Arnold, A., & Pestell, R. G. (2015). Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis. Oncotarget, 6(11), 8525-38.More infoCyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.
- Hutcheson, J., Witkiewicz, A. K., & Knudsen, E. S. (2015). The RB tumor suppressor at the intersection of proliferation and immunity: relevance to disease immune evasion and immunotherapy. Cell cycle (Georgetown, Tex.), 14(24), 3812-9.More infoThe retinoblastoma tumor suppressor (RB) was the first identified tumor suppressor based on germline predisposition to the pediatric eye tumor. Since these early studies, it has become apparent that the functional inactivation of RB is a common event in nearly all human malignancy. A great deal of research has gone into understanding how the loss of RB promotes tumor etiology and progression. Since malignant tumors are characterized by aberrant cell division, much of this research has focused upon the ability of RB to regulate the cell cycle by repression of proliferation-related genes. However, it is progressively understood that RB is an important mediator of multiple functions. One area that is gaining progressive interest is the emerging role for RB in regulating diverse features of immune function. These findings suggest that RB is more than simply a regulator of cellular proliferation; it is at the crossroads of proliferation and the immune response. Here we review the data related to the functional roles of RB on the immune system, relevance to immune evasion, and potential significance to the response to immune-therapy.
- Knudsen, E. S., McClendon, A. K., Franco, J., Ertel, A., Fortina, P., & Witkiewicz, A. K. (2015). RB loss contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer. Cell cycle (Georgetown, Tex.), 14(1), 109-22.More infoTriple negative breast cancer (TNBC) is characterized by multiple genetic events occurring in concert to drive pathogenic features of the disease. Here we interrogated the coordinate impact of p53, RB, and MYC in a genetic model of TNBC, in parallel with the analysis of clinical specimens. Primary mouse mammary epithelial cells (mMEC) with defined genetic features were used to delineate the combined action of RB and/or p53 in the genesis of TNBC. In this context, the deletion of either RB or p53 alone and in combination increased the proliferation of mMEC; however, the cells did not have the capacity to invade in matrigel. Gene expression profiling revealed that loss of each tumor suppressor has effects related to proliferation, but RB loss in particular leads to alterations in gene expression associated with the epithelial-to-mesenchymal transition. The overexpression of MYC in combination with p53 loss or combined RB/p53 loss drove rapid cell growth. While the effects of MYC overexpression had a dominant impact on gene expression, loss of RB further enhanced the deregulation of a gene expression signature associated with invasion. Specific RB loss lead to enhanced invasion in boyden chambers assays and gave rise to tumors with minimal epithelial characteristics relative to RB-proficient models. Therapeutic screening revealed that RB-deficient cells were particularly resistant to agents targeting PI3K and MEK pathway. Consistent with the aggressive behavior of the preclinical models of MYC overexpression and RB loss, human TNBC tumors that express high levels of MYC and are devoid of RB have a particularly poor outcome. Together these results underscore the potency of tumor suppressor pathways in specifying the biology of breast cancer. Further, they demonstrate that MYC overexpression in concert with RB can promote a particularly aggressive form of TNBC.
- Witkiewicz, A. K., Borja, N. A., Franco, J., Brody, J. R., Yeo, C. J., Mansour, J., Choti, M. A., McCue, P., & Knudsen, E. S. (2015). Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer. Oncotarget, 6(18), 15788-801.More infoPancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities.
- Witkiewicz, A. K., McMillan, E. A., Balaji, U., Baek, G., Lin, W., Mansour, J., Mollaee, M., Wagner, K., Koduru, P., Yopp, A., Choti, M. A., Yeo, C. J., McCue, P., White, M. A., & Knudsen, E. S. (2015). Whole-exome sequencing of pancreatic cancer defines genetic diversity and therapeutic targets. Nature communications, 6, 6744.More infoPancreatic ductal adenocarcinoma (PDA) has a dismal prognosis and insights into both disease etiology and targeted intervention are needed. A total of 109 micro-dissected PDA cases were subjected to whole-exome sequencing. Microdissection enriches tumour cellularity and enhances mutation calling. Here we show that environmental stress and alterations in DNA repair genes associate with distinct mutation spectra. Copy number alterations target multiple tumour suppressive/oncogenic loci; however, amplification of MYC is uniquely associated with poor outcome and adenosquamous subtype. We identify multiple novel mutated genes in PDA, with select genes harbouring prognostic significance. RBM10 mutations associate with longer survival in spite of histological features of aggressive disease. KRAS mutations are observed in >90% of cases, but codon Q61 alleles are selectively associated with improved survival. Oncogenic BRAF mutations are mutually exclusive with KRAS and define sensitivity to vemurafenib in PDA models. High-frequency alterations in Wnt signalling, chromatin remodelling, Hedgehog signalling, DNA repair and cell cycle processes are observed. Together, these data delineate new genetic diversity of PDA and provide insights into prognostic determinants and therapeutic targets.
- Baek, G., Tse, Y. F., Hu, Z., Cox, D., Buboltz, N., McCue, P., Yeo, C. J., White, M. A., DeBerardinis, R. J., Knudsen, E. S., & Witkiewicz, A. K. (2014). MCT4 defines a glycolytic subtype of pancreatic cancer with poor prognosis and unique metabolic dependencies. Cell reports, 9(6), 2233-49.More infoKRAS mutation, which occurs in ∼ 95% of pancreatic ductal adenocarcinoma (PDA), has been shown to program tumor metabolism. MCT4 is highly upregulated in a subset of PDA with a glycolytic gene expression program and poor survival. Models with high levels of MCT4 preferentially employ glycolytic metabolism. Selectively in such "addicted" models, MCT4 attenuation compromised glycolytic flux with compensatory induction of oxidative phosphorylation and scavenging of metabolites by macropinocytosis and autophagy. In spite of these adaptations, MCT4 depletion induced cell death characterized by elevated reactive oxygen species and metabolic crisis. Cell death induced by MCT4-depletion was augmented by inhibition of compensatory pathways. In xenograft models, MCT4 had a significant impact on tumor metabolism and was required for rapid tumor growth. Together, these findings illustrate the metabolic diversity of PDA described by MCT4, delineate pathways through which this lactate transporter supports cancer growth, and demonstrate that PDA can be rationally targeted based on metabolic addictions.
- Franco, J., Witkiewicz, A. K., & Knudsen, E. S. (2014). CDK4/6 inhibitors have potent activity in combination with pathway selective therapeutic agents in models of pancreatic cancer. Oncotarget, 5(15), 6512-25.More infoPancreatic ductal adenocarcinoma (PDA) has a poor prognosis, in part, due to the therapy-recalcitrant nature of the disease. Loss of the CDK4/6 inhibitor CDKN2A is a signature genetic event in PDA. Therefore, PDA may be amenable to treatment with pharmaceutical CDK4/6 inhibitors. Surprisingly, response to CDK4/6 inhibition was highly variable in PDA models, and associated with differential suppression of gene expression. Mitotic genes were repressed and FOXM1 was uniformly attenuated; however, genes involved in DNA replication were uniquely suppressed in sensitive models. Aberrant induction of Cyclin E1 was associated with resistance, and knockdown demonstrated synergistic suppression of the cell cycle with CDK4/6 inhibition. Combination therapies are likely required for the effective treatment of disease, and drug screening demonstrated additive/antagonistic interactions with CDK4/6 inhibitors. Agents dependent on mitotic progression (taxanes/PLK1 inhibitors) were antagonized by CDK4/6 inhibition, while the response to 5-FU and gemcitabine exhibited drug specific interactions. PI3K/MTOR and MEK inhibitors potently cooperated with CDK4/6 inhibition. These agents were synergistic with CDK4/6 inhibition, blocked the aberrant upregulation of Cyclin E1, and yielded potent inhibition of tumor cell growth. Together, these data identify novel mechanisms of resistance to CDK4/6 inhibitions and provide a roadmap for combination therapies in the treatment of PDA.
- Hutcheson, J., Bourgo, R. J., Balaji, U., Ertel, A., Witkiewicz, A. K., & Knudsen, E. S. (2014). Retinoblastoma protein potentiates the innate immune response in hepatocytes: significance for hepatocellular carcinoma. Hepatology (Baltimore, Md.), 60(4), 1231-40.More infoCancers mediated by viral etiology must exhibit deregulated cellular proliferation and evade immune recognition. The role of the retinoblastoma tumor suppressor (RB) pathway, which is lost at relatively high frequency in hepatocellular carcinoma (HCC), has recently been expanded to include the regulation of innate immune responsiveness. In this study we investigated the coordinate impact of RB-loss on cell cycle control and immune function in the liver. We found that RB depletion in hepatoma cells resulted in a compromised immunological response to multiple stimuli and reduced the potential of these cells to recruit myeloid cells. Viral-mediated liver-specific RB deletion in vivo led to the induction of genes associated with proliferation and cell cycle entry as well as the significant attenuation of genes associated with immune function, as evidenced by decreases in cytokine and chemokine expression, leukocyte recruitment, and hepatic inflammation. To determine if these changes in gene expression were instructive in human disease, we compared our liver-specific RB-loss gene signature to existing profiles of HCC and found that this signature was associated with disease progression and confers a worse prognosis.
- Knudsen, E. S., Gopal, P., & Singal, A. G. (2014). The changing landscape of hepatocellular carcinoma: etiology, genetics, and therapy. The American journal of pathology, 184(3), 574-83.More infoHepatocellular carcinoma (HCC) represents one of the leading causes of cancer death and has proved to be highly refractory to treatment. Extensive analysis of the disease has demonstrated that it arises predominantly in response to high-risk etiological challenges, most notably hepatitis virus. However, with evolving vaccination and the obesity epidemic, progressively more cases are associated with underlying metabolic dysfunction. Pathologically diverse forms of HCC are observed, and recent sequencing analysis has defined common events that target well-known cancer pathways including β-catenin/Axin, TP53, and RB/CDKN2A, as well as frequent aberrations in chromatin remodeling factors. However, there are a myriad of low frequency genetic events that make each HCC case unique. Gene expression profiling approaches have successfully been deployed for prognostic assessment of hepatocellular carcinoma and to detect the earliest stages of disease. Despite more extensive research, systemic treatment for HCC is exceedingly limited, with only a handful of drugs providing benefit. Ongoing clinical trials are attempting to exploit specific biological dependencies of HCC to improve the dismal prognosis. Overall, the future of HCC treatment will rely on an understanding of the interplay between etiological factors, molecular features of disease, and rational therapeutic intervention.
- Reed, C., Hutcheson, J., Mayhew, C. N., Witkiewicz, A. K., & Knudsen, E. S. (2014). RB tumor suppressive function in response to xenobiotic hepatocarcinogens. The American journal of pathology, 184(6), 1853-9.More infoDiverse etiologic events are associated with the development of hepatocellular carcinoma. During hepatocarcinogenesis, genetic events likely occur that subsequently cooperate with long-term exposures to further drive the progression of hepatocellular carcinoma. In this study, the frequent loss of the retinoblastoma (RB) tumor suppressor in hepatocellular carcinoma was modeled in response to diverse hepatic stresses. Loss of RB did not significantly affect the response to a steatotic stress as driven by a methionine- and choline-deficient diet. In addition, RB status did not significantly influence the response to peroxisome proliferators that can drive hepatomegaly and tumor development in rodents. However, RB loss exhibited a highly significant effect on the response to the xenobiotic1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene. Loss of RB yielded a unique proliferative response to this agent, which was distinct from both regenerative stresses and genotoxic carcinogens. Long-term exposure to 1,4-Bis-[2-(3,5-dichloropyridyloxy)] benzene yielded profound tumor development in RB-deficient livers that was principally absent in RB-sufficient tissue. These data demonstrate the context specificity of RB and the key role RB plays in the suppression of hepatocellular carcinoma driven by xenobiotic stress.
- Witkiewicz, A. K., & Knudsen, E. S. (2014). Retinoblastoma tumor suppressor pathway in breast cancer: prognosis, precision medicine, and therapeutic interventions. Breast cancer research : BCR, 16(3), 207.More infoA series of recent studies have demonstrated that the retinoblastoma tumor suppressor (RB) pathway plays a critical role in multiple clinically relevant aspects of breast cancer biology, spanning early stage lesions to targeted treatment of metastatic disease. In ductal carcinoma in situ, multiple groups have shown that dysregulation of the RB pathway is critically associated with recurrence and disease progression. Functional models have similarly illustrated key roles for RB in regulating epithelial-mesenchymal transition and other features contributing to aggressive disease. Invasive breast cancers are treated in distinct fashions, and heterogeneity within the RB pathway relates to prognosis and response to commonly used therapeutics. Luminal B breast cancers that have a poor prognosis amongst estrogen receptor-positive disease are defined based on the expression of RB-regulated genes. Such findings have led to clinical interventions that directly target the RB pathway through CDK4/6 inhibition which have promise in both estrogen receptor-positive and Her2-positive disease. In contrast, RB loss results in improved response to chemotherapy in triple-negative breast cancer, where ongoing research is attempting to define intrinsic vulnerabilities for targeted intervention. These findings support a wide-reaching impact of the RB pathway on disease that could be harnessed for improved clinical interventions.
- Witkiewicz, A. K., Balaji, U., & Knudsen, E. S. (2014). Systematically defining single-gene determinants of response to neoadjuvant chemotherapy reveals specific biomarkers. Clinical cancer research : an official journal of the American Association for Cancer Research, 20(18), 4837-48.More infoWe sought to systematically define determinants of the response to neoadjuvant chemotherapy to elucidate predictive biomarkers for breast cancer.
- Witkiewicz, A. K., Cox, D., & Knudsen, E. S. (2014). CDK4/6 inhibition provides a potent adjunct to Her2-targeted therapies in preclinical breast cancer models. Genes & cancer, 5(7-8), 261-72.More infoIn spite of the efficacy of Her2-targeted therapies, recurrence and progression remain a challenge for treatment of Her2 positive breast cancer. CDK4/6 controls pathway downstream of Her2, Inhibition of these kinases could represent an important therapeutic approach to augment the effectiveness of standard therapies. In models of acquired resistance to Her2-targeted therapies, Cyclin D1 was inappropriately activated and CDK4/6 inhibition was effective at blocking proliferation by targeting this common pathway associated with resistance. These data were recapitulated in Her2 positive xenografts. Furthermore, in a series of 35 primary breast tumor explants, treatment with PD-0332991 resulted in a greater than 4-fold suppression of the Ki67. The effects of CDK4/6 inhibition were dependent on an intact RB-pathway, and consonantly, loss of RB and high-levels of p16 were associated with resistance to CDK4/6 inhibition. Combination studies illustrated that CDK4/6 inhibition is cooperative with multiple Her2-targeted agents and provides a complementary mechanism of action to T-DM1 to efficiently suppresses the proliferation of residual Her2-positive tumor cell populations that survive T-DM1. Together, these data indicate CDK4/6 is a viable therapeutic target that functions downstream of Her2, and tissue based markers are available to direct rational utilization of CDK4/6 inhibitors in combination with Her2-targeted agents.
- Knudsen, E. S., Dervishaj, O., Kleer, C. G., Pajak, T., Schwartz, G. F., & Witkiewicz, A. K. (2013). EZH2 and ALDH1 expression in ductal carcinoma in situ: complex association with recurrence and progression to invasive breast cancer. Cell cycle (Georgetown, Tex.), 12(13), 2042-50.More infoThe diagnosis of ductal carcinoma in situ (DCIS) is an increasingly common event due to widespread use of screening mammography. However, appropriate clinical management of DCIS is a major challenge in the absence of prognostic markers. Tumor-initiating cells may be particularly relevant for disease pathogenesis; therefore, two markers associated with such cells, EZH2 and ALDH1, were evaluated. A cohort of 248 DCIS patients was used to determine the association of EZH2 and ALDH1 with ipsilateral breast event, DCIS recurrence and progression to invasive breast cancer (IBC). In this cohort, high EZH2 expression was associated with the risk of an ipsilateral breast event and DCIS recurrence but not invasive progression. ALDH1 expression was observed in both the tumor and stromal compartment; however, in neither compartment were ALDH1 levels independently associated with evaluated study endpoints. Interestingly, the combination of high EZH2 with high epithelial ALDH1 was associated with disease progression. Therefore, ALDH1 within the DCIS lesion can add to the prognostic significance of EZH2, particularly in the context of risk of development of invasive disease.
- Thangavel, C., Boopathi, E., Ertel, A., Lim, M., Addya, S., Fortina, P., Witkiewicz, A. K., & Knudsen, E. S. (2013). Regulation of miR106b cluster through the RB pathway: mechanism and functional targets. Cell cycle (Georgetown, Tex.), 12(1), 98-111.More infoThe RB pathway plays a critical role in proliferation control that is commonly subverted in tumor development. However, restoration of RB pathway function can be elicited in many tumor cells by the inhibition of CDK4/6 activity that leads to dephosphorylation of RB and subsequent repression of E2F-mediated transcription. In this context, active RB/E2F complexes inhibit the expression of a critical program of coding genes that promote cell cycle progression. However, the non-coding RNA target genes downstream from RB that could be relevant for tumor biology remain obscure. Here, miRNA gene expression profiling identified the miR106b cluster as being efficiently repressed with CDK4/6 inhibition in an E2F and RB-dependent manner. Importantly, the miR106B-cluster is intragenic of MCM7, and through a series of functional studies, the basis of MCM7 regulation and concordant expression of the miRNA species within the 106b cluster was determined. Importantly, RB-mediated repression of the 106b cluster enhances the transcript levels of p21Cip1 and PTEN. These data provide a mechanistic basis for cross-talk between the RB pathway and p21 and PTEN through the regulation of the MCM7/miR106b locus.
- Casimiro, M. C., Crosariol, M., Loro, E., Ertel, A., Yu, Z., Dampier, W., Saria, E. A., Papanikolaou, A., Stanek, T. J., Li, Z., Wang, C., Fortina, P., Addya, S., Tozeren, A., Knudsen, E. S., Arnold, A., & Pestell, R. G. (2012). ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice. The Journal of clinical investigation, 122(3), 833-43.More infoChromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1(-/-) mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.
- Dean, J. L., McClendon, A. K., & Knudsen, E. S. (2012). Modification of the DNA damage response by therapeutic CDK4/6 inhibition. The Journal of biological chemistry, 287(34), 29075-87.More infoThe RB/E2F axis represents a critical node of cell signaling that integrates a diverse array of signaling pathways. Recent evidence has suggested a role for E2F-mediated gene transcription in DNA damage response and repair, as well as apoptosis signaling. Herein, we investigated how repression of E2F activity via CDK4/6 inhibition and RB activation impacts the response of triple negative breast cancer (TNBC) to frequently used therapeutic agents. In combination with taxanes and anthracyclines CDK4/6 inhibition and consequent cell cycle arrest prevented the induction of DNA damage and associated cell death in an RB-dependent manner; thereby demonstrating antagonism between the cytostatic influence of the CDK-inhibitor and cytotoxic agents. As many of these effects were secondary to cell cycle arrest, γ-irradiation (IR) was utilized to examine effects of CDK4/6 inhibition on direct DNA damage. Although E2F controls a number of genes involved in DNA repair (e.g. Rad51), CDK4/6 inhibition did not alter the overall rate of DNA repair, rather it significantly shifted the burden of this repair from homologous recombination (HR) to non-homologous end joining (NHEJ). Together, these data indicate that CDK4/6 inhibition can antagonize cytotoxic therapeutic strategies and increases utilization of error-prone DNA repair mechanisms that could contribute to disease progression.
- Dean, J. L., McClendon, A. K., Hickey, T. E., Butler, L. M., Tilley, W. D., Witkiewicz, A. K., & Knudsen, E. S. (2012). Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors. Cell cycle (Georgetown, Tex.), 11(14), 2756-61.More infoTo model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.
- Knudsen, E. S., Ertel, A., Davicioni, E., Kline, J., Schwartz, G. F., & Witkiewicz, A. K. (2012). Progression of ductal carcinoma in situ to invasive breast cancer is associated with gene expression programs of EMT and myoepithelia. Breast cancer research and treatment, 133(3), 1009-24.More infoDuctal carcinoma in situ (DCIS) is a precursor lesion that can gives rise to invasive breast cancer (IBC). It has been proposed that both the nature of the lesion and the tumor microenvironment play key roles in progression to IBC. Here, laser capture microdissected tissue from pure DCIS and pure IBC were employed to define key gene expression profiles in either the epithelial or stromal compartment associated with disease progression. Each tissue had distinct gene expression profiles, and a DCIS/IBC classifier accurately distinguished DCIS versus IBC in multiple independent data sets. However, contrary to other studies that profiled DCIS associated with invasive disease, we found that the most significant alterations in gene expression were observed in the epithelial compartment rather than in the stroma. In particular, genes associated with epithelial-to-mesenchymal transition and myoepithelial cell-specific genes were enriched in invasive cancer relative to pure DCIS. Such alterations in transcript levels were associated with all subtypes of breast cancer, but were particularly indicative of poor outcome in ER-negative breast cancer. Together, these studies indicate that lesion-specific differences in gene expression associated with invasive phenotype are particularly relevant in the progression of DCIS to invasive breast cancer.
- Knudsen, E. S., Pajak, T. F., Qeenan, M., McClendon, A. K., Armon, B. D., Schwartz, G. F., & Witkiewicz, A. K. (2012). Retinoblastoma and phosphate and tensin homolog tumor suppressors: impact on ductal carcinoma in situ progression. Journal of the National Cancer Institute, 104(23), 1825-36.More infoA subset of patients with ductal carcinoma in situ (DCIS) will progress to invasive breast cancer. However, there are currently no markers to differentiate women at high risk from those at lower risk of developing invasive disease.
- McClendon, A. K., Dean, J. L., Rivadeneira, D. B., Yu, J. E., Reed, C. A., Gao, E., Farber, J. L., Force, T., Koch, W. J., & Knudsen, E. S. (2012). CDK4/6 inhibition antagonizes the cytotoxic response to anthracycline therapy. Cell cycle (Georgetown, Tex.), 11(14), 2747-55.More infoTriple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.
- Witkiewicz, A. K., Ertel, A., McFalls, J., Valsecchi, M. E., Schwartz, G., & Knudsen, E. S. (2012). RB-pathway disruption is associated with improved response to neoadjuvant chemotherapy in breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 18(18), 5110-22.More infoWe sought to determine whether dysregulation of the retinoblastoma (RB) tumor suppressor pathway was associated with improved response to neoadjuvant chemotherapy in breast cancer.
- Bourgo, R. J., Thangavel, C., Ertel, A., Bergseid, J., McClendon, A. K., Wilkens, L., Witkiewicz, A. K., Wang, J. Y., & Knudsen, E. S. (2011). RB restricts DNA damage-initiated tumorigenesis through an LXCXE-dependent mechanism of transcriptional control. Molecular cell, 43(4), 663-72.More infoThe LXCXE peptide motif facilitates interaction between the RB tumor suppressor and a large number of cellular proteins that are expected to impinge on diverse biological processes. In vitro and in vivo analyses demonstrated that LXCXE binding function is dispensable for RB promoter association and control of basal gene expression. Dependence on this function of RB is unmasked after DNA damage, wherein LXCXE binding is essential for exerting control over E2F3 and suppressing cell-cycle progression in the presence of genotoxic stress. Gene expression profiling revealed that the transcriptional program coordinated by this specific aspect of RB is associated with progression of human hepatocellular carcinoma and poor disease outcome. Consistent with these findings, biological challenge revealed a requirement for LXCXE binding in suppression of genotoxin-initiated hepatocellular carcinoma in vivo. Together, these studies establish an essential role of the LXCXE binding motif for RB-mediated transcriptional control, response to genotoxic insult, and tumor suppression.
- McClendon, A. K., Dean, J. L., Ertel, A., Fu, Z., Rivadeneira, D. B., Reed, C. A., Bourgo, R. J., Witkiewicz, A., Addya, S., Mayhew, C. N., Grimes, H. L., Fortina, P., & Knudsen, E. S. (2011). RB and p53 cooperate to prevent liver tumorigenesis in response to tissue damage. Gastroenterology, 141(4), 1439-50.More infoThe tumor suppressors retinoblastoma (RB) and p53 are important regulators of the cell cycle. Although human cancer cells inactivate RB and p53 by many mechanisms, the cooperative roles of these proteins in tumorigenesis are complex and tissue specific. We analyzed the cooperation of RB and p53 in liver development and pathogenesis of hepatocellular carcinoma.
- Thangavel, C., Dean, J. L., Ertel, A., Knudsen, K. E., Aldaz, C. M., Witkiewicz, A. K., Clarke, R., & Knudsen, E. S. (2011). Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer. Endocrine-related cancer, 18(3), 333-45.More infoThe majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.
- Witkiewicz, A. K., & Knudsen, E. S. (2011). RB pathway and therapeutic sensitivity: new insights in breast cancer and Tamoxifen therapy. Cell cycle (Georgetown, Tex.), 10(10), 1525.
- Witkiewicz, A. K., Knudsen, K. E., Dicker, A. P., & Knudsen, E. S. (2011). The meaning of p16(ink4a) expression in tumors: functional significance, clinical associations and future developments. Cell cycle (Georgetown, Tex.), 10(15), 2497-503.More infoThe CDKN2A gene is a tumor suppressor that encodes the CDK4/6 inhibitor p16(ink4a). Loss of this tumor suppressor contributes to the bypass of critical senescent signals and is associated with progression to malignant disease. However, the high-level expression of p16(ink4a) in tumors is associated with aggressive subtypes of disease, and in certain clinical settings elevated p16(ink4a) expression is an important determinant for disease prognosis and therapeutic response. These seemingly contradictory facets of p16(ink4a) expression have lead to confusion related to the meaning of this tumor suppression in tumor pathobiology. As reviewed here, the alternative expression of p16(ink4a) represents an ideal marker for considering RB-pathway function, tumor heterogeneity, and novel means for directing therapy.
- Witkiewicz, A. K., Rivadeneira, D. B., Ertel, A., Kline, J., Hyslop, T., Schwartz, G. F., Fortina, P., & Knudsen, E. S. (2011). Association of RB/p16-pathway perturbations with DCIS recurrence: dependence on tumor versus tissue microenvironment. The American journal of pathology, 179(3), 1171-8.More infoThe prevalence of ductal carcinoma in situ (DCIS) diagnoses has significantly increased as a result of active radiographic screening. Surgical resection and hormone and radiation therapies are effective treatments, but not all DCIS will progress to invasive breast cancer. Therefore, markers are needed to define tumors at low risk of recurrence and progression that can be treated by surgery alone rather than by adjuvant therapies. Initial analyses indicate that retinoblastoma (RB)-pathway perturbations occur at high frequency in DCIS and mirror the molecular alterations observed in invasive breast cancer. Particularly, the elevated expression of p16ink4a in DCIS was associated with loss of RB function and estrogen receptor-negative biology. Furthermore, high expression of p16ink4a in conjunction with Ki-67 was associated with increased risk of DCIS recurrence and progression to invasive disease in multivariate analyses. These data are consistent with a functional role for RB in modulating the invasive behavior of mammary epithelial cells. The tissue microenvironment is particularly relevant to the behavior of DCIS, and, surprisingly, elevated expression of p16ink4a in nonproliferative stroma was observed in a substantial fraction of cases. In this tissue compartment, p16ink4a expression was strongly associated with disease recurrence, independent of standard histopathologic features. Together, the data herein describe dual aspects of RB-pathway biology that are associated with disease recurrence through the epithelial or stromal compartment of DCIS.
- Ertel, A., Dean, J. L., Rui, H., Liu, C., Witkiewicz, A. K., Knudsen, K. E., & Knudsen, E. S. (2010). RB-pathway disruption in breast cancer: differential association with disease subtypes, disease-specific prognosis and therapeutic response. Cell cycle (Georgetown, Tex.), 9(20), 4153-63.More infoIn breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.
- Martinez-Outschoorn, U. E., Balliet, R. M., Rivadeneira, D. B., Chiavarina, B., Pavlides, S., Wang, C., Whitaker-Menezes, D., Daumer, K. M., Lin, Z., Witkiewicz, A. K., Flomenberg, N., Howell, A., Pestell, R. G., Knudsen, E. S., Sotgia, F., & Lisanti, M. P. (2010). Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells. Cell cycle (Georgetown, Tex.), 9(16), 3256-76.More infoLoss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a "lethal tumor micro-environment." Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a "metabolic" and "mutagenic" motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the "Reverse Warburg Effect"). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use "oxidative stress" in adjacent fibroblasts (i) as an "engine" to fuel their own survival via the stromal production of nutrients and (ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the "field effect" in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to downregulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified and are effectively "contagious"--spread from cell-to-cell like a virus--creating an "oncogenic/mutagenic" field promoting widespread DNA damage.
- Sharma, A., Yeow, W. S., Ertel, A., Coleman, I., Clegg, N., Thangavel, C., Morrissey, C., Zhang, X., Comstock, C. E., Witkiewicz, A. K., Gomella, L., Knudsen, E. S., Nelson, P. S., & Knudsen, K. E. (2010). The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression. The Journal of clinical investigation, 120(12), 4478-92.More infoRetinoblastoma (RB; encoded by RB1) is a tumor suppressor that is frequently disrupted in tumorigenesis and acts in multiple cell types to suppress cell cycle progression. The role of RB in tumor progression, however, is poorly defined. Here, we have identified a critical role for RB in protecting against tumor progression through regulation of targets distinct from cell cycle control. In analyses of human prostate cancer samples, RB loss was infrequently observed in primary disease and was predominantly associated with transition to the incurable, castration-resistant state. Further analyses revealed that loss of the RB1 locus may be a major mechanism of RB disruption and that loss of RB function was associated with poor clinical outcome. Modeling of RB dysfunction in vitro and in vivo revealed that RB controlled nuclear receptor networks critical for tumor progression and that it did so via E2F transcription factor 1-mediated regulation of androgen receptor (AR) expression and output. Through this pathway, RB depletion induced unchecked AR activity that underpinned therapeutic bypass and tumor progression. In agreement with these findings, disruption of the RB/E2F/nuclear receptor axis was frequently observed in the transition to therapy resistance in human disease. Together, these data reveal what we believe to be a new paradigm for RB function in controlling prostate tumor progression and lethal tumor phenotypes.