Tumor phosphatidylinositol 3-kinase signaling in therapy resistance and metastatic dissemination of rectal cancer: Opportunities for signaling-adapted therapies

https://doi.org/10.1016/j.critrevonc.2015.01.003Get rights and content

Highlights

  • Biomarkers of treatment failure were quested in a prospective rectal cancer study.

  • A kinase substrate array technology was applied to analyze patients’ tumor samples.

  • High tumor PI3K signaling was correlated with adverse survival outcome.

  • The specific tumor signature of PI3K signaling points to actionable therapy targets.

Abstract

Locally advanced rectal cancer (LARC) comprises heterogeneous tumors with predominant hypoxic components, a hallmark of the tumor microenvironment and determinant of resistance to cytotoxic therapies, local recurrence, and metastatic progression. A rational integration of molecularly targeted agents in established combined-modality treatment regimens may improve local and systemic disease control, but will require a clear definition of functional biomarkers for patient stratification. In a prospective study of LARC patients given neoadjuvant chemotherapy and radiation, we applied a kinase substrate array technology to analyze the patients’ tumor biopsies sampled at the time of diagnosis, and observed that receptor tyrosine kinase activities integrated by high phosphatidylinositol 3-kinase signaling were correlated both with poor tumor response to the neoadjuvant treatment and adverse progression-free survival. Conceptually, the specific tumor signature of phosphatidylinositol 3-kinase signaling activity may point to actionable therapy targets in LARC patients with unfavorable disease features. Clinical trial registration number NCT00278694.

Introduction

With reference to the demography of colorectal cancer (CRC), owing to an aging population, this disease is common with a significant rise in incidence from the age of 60. In the European Union in 2012, more than 447,000 individuals were diagnosed with CRC; of these cases, about 30% were in the rectal anatomic site [1]. The management of rectal cancer is multidisciplinary, involving precision diagnostics within radiology and pathology and highly specialized expertise within oncology and surgery. Recognizing the therapeutic complexities, it is crucial to bridge the rapidly emerging knowledge from biology into the optimum care of patients.

As a result of systematic improvements that include multimodal therapy, primarily surgery and radiation, over the past few decades, long-term control of localized rectal cancer is commonly achieved. Locally advanced rectal cancer (LARC) comprises primary tumors that infiltrate within the mesorectal compartment or beyond to an extent that precludes primary surgical removal with sufficient microscopic margins. Randomized studies have highlighted the central role of neoadjuvant chemoradiotherapy (CRT) in macroscopic down-sizing and control of subclinical tumor manifestations within the pelvic cavity, to enable complete resection of the residual tumor within its entire extension for the ultimate improvement of outcome [2]. There is compelling evidence from large cohorts of patients given neoadjuvant CRT that histologic tumor down-staging translates into long-term survival benefits; however, on a closer look, this observation basically refers to subgroups of patients obtaining complete tumor response [3], suggesting that eradication of tumor clonogens is essential for favorable therapeutic results.

Even with successful local treatment outcome, a substantial number of LARC patients (30–40% of cases) will experience metastatic progression [4], [5] as result of distant organ establishment of early disseminating tumor cells (DTC) [6], [7] with clonogenic potential. Currently, no consensus exists to whether systemic therapy may reduce the risk of metastatic development in rectal cancer [5], [8]. The use of postoperative adjuvant chemotherapy, as being given in colon cancer, has been adopted at a number of centers internationally despite the lack of evidence-based data [9]. In most Nordic countries, postoperative treatment is offered only on specific, individual-based indications.

Metastatic disease may be either organ-confined or extensively disseminated to the liver, lungs, peritoneum, multiple lymph node stations and skeletal locations, or even the brain. Metastasis limited to a single organ is increasingly considered potentially curable with multimodal approaches. Examples include disease confined to the liver [10], lungs [11], or the peritoneal cavity [12]. New insights into the biology of early systemic tumor dissemination may provide a direction to the next milestone in rectal cancer management, which will be the control of metastatic progression.

In line with these notions, and in the everyday clinical practice, it is recognized that rectal cancer presents with a high degree of biological heterogeneity [13]. The prevailing hypothesis is that in the primary tumor, expansion of distinct subclones under selection pressure within specific microenvironmental niches gives rise to such heterogeneity [14]. Consequently, a critical understanding of the biological diversity is needed to improve existing therapies both for the initial eradication of primary tumor growth and importantly, prevention of metastatic progression.

Considerable interest is focused on the specific characteristics of the tumor microenvironment, of which hypoxia is one of the most important hallmarks [15], [16], [17]. The hypoxic phenotype is recognized as a main mechanism in tumor resistance to cytotoxic therapy (radiation and chemotherapy) [18], [19] and is significantly correlated to tumor recurrence and metastatic disease progression [20], [21].

At the molecular level, hypoxia is a driver of tumor metabolism and promotes angiogenesis, metastasis, and therapy resistance through the alteration of oxygen-sensitive regulatory mechanisms [16], [22]. The adaptive tumor responses to hypoxic stress involve intrinsic activation of a range of signaling pathways mediated by receptor tyrosine kinases such as EGFR, VEGFR, and PDGFR family members, collectively contributing to the continuous augmentation of the malignant phenotype [17].

The multitude of more than 500 protein kinases, the kinome, acts as key regulators of cell function by catalyzing the addition of phosphate groups to target proteins within the signal transduction cascade. In normal physiological responses, these phosphorylation processes are tightly controlled [23]. As much as half of the tyrosine kinase complement is implicated in cancer, and specifically, a number of tyrosine kinases will gain oncogenic capabilities following aberrations in their encoding genes [24]. All of this has led to the recognition of receptor tyrosine kinase-governed signaling pathways as potential biomarkers for stratification of patients to molecularly individualized therapies. Moreover, the significance of tumor kinase activities is reflected by the extensive pharmaceutical development of targeted therapeutics.

Radiotherapy is an effective treatment for cancer when the disease presents at an early stage; however, radiation may fail to eradicate all of the clonogenic cells within a heterogeneous tumor. The tangible reality of this failure is local or distant disease recurrence. With current advances in molecular radiobiology, strategies are increasingly focused on inhibiting tumor cell signaling to augment the DNA damage-induced cytotoxicity [18], [25]. Within this frame of reference, an emerging clinical concept is demonstrated by the escalating number of radiotherapy studies reporting on the use of systemic targeted agents toward receptor tyrosine kinase-directed signaling as radiation sensitizers [26], [27].

Under normal physiological conditions, the mammalian target of rapamycin (mTOR) complex-1, with its master player mTOR, is a molecular hub integrating inhibitory signaling activities of cellular stress responses to hypoxia and DNA damage. Major input pathways are routed through phosphatidylinositol 3-kinase (PI3K) and AKT, mediating a range of receptor tyrosine kinase activities [28], [29]. In the context of dysregulated tumor signaling, however, these effector mechanisms are constitutively activated and reflect adaptation responses to promote tumor cell survival within a hostile hypoxic microenvironment [29], [30].

The intense focus on molecularly targeted compounds, both as single-agent therapy and for optimization of multimodality cancer treatment, has been facilitated by the tremendous developments in high-throughput comprehensive technologies for molecular profiling. The currently most developed technologies enable determination of genetic (gene mutation, copy number, or translocation) and genomic (gene expression profiles) biomarkers [31]. In selecting patients to molecularly targeted agents, when a validated biomarker exists, the prevailing gold standards are mainly based on detection of tumor gene aberrations embodied as the absence or presence of specific mutations, the latter being activating or inhibiting, or as amplifications or translocations [32]. However, such biomarkers may not be sufficient for the purpose since multiple gene aberrations, which in solid tumors often are the case rather than a single driving gene modification, will affect a wide range of components of the signaling network. Of further note, the plasticity of microenvironmental changes in hypoxic tumors will also contribute to the diversity in signaling activity.

Consequently, with the complexity of kinase signaling reflecting the multiplicity of specific gene mutations while other aberrations may not influence kinase integrity, methodologies comprising the resultant condition of interacting signaling effects may be particularly advantageous to identify functional biomarkers for targeted agents. However, until recently, large-scale profiling of tissue kinases has been hindered by the unavailability of feasible methods in clinical practice. Established phosphoproteomic technologies, such as mass spectrometry – the gold standard within the field, enable measurement of a very high number of phosphorylation sites within the proteome. But many of these methodologies are generally time-consuming and require large tissue quantities, which will preclude applicability for diagnostic purposes. Ideally, to become clinically useful, technologies must provide rapid and high-fidelity analysis of small-quantity samples. Kinase substrate array technologies are high-throughput tools for global profiling of kinase activities in tissue samples without prior knowledge of which signaling pathways are activated [33], [34], [35], [36]. Using such arrays, each tumor sample will generate an individual phosphosubstrate signature, theoretically portraying the state of composite information flow through signaling cascades.

As outlined in Fig. 1, the format of this assay (PamGene International B.V., ’s-Hertogenbosch, The Netherlands), which we have applied to study tumor biopsies from patients with rectal and prostate cancer and malignant melanoma [37], [38], [39], [40], [41], is an array containing 144 peptides. Each of these kinase targets consists of 13 or 14 amino acids with tyrosine residues for phosphorylation (Supplementary Table S1). Substrate phosphorylation levels are assessed from a bound fluorescent anti-phosphotyrosine antibody. The technology is robust with small tissue quantities, typically 5–15 μg of total protein being sufficient. To provide additional information of specific signaling pathways that mechanistically may be important for the biological process of investigation, the tissue lysate can be incubated on the array also in the presence of a selected small-molecular kinase inhibitor for measurement of specific alterations in substrate phosphorylation levels. In this manner, the ex vivo substrate specificity of the kinase-inhibitory agent may also indicate signaling mechanisms that potentially may be actionable tumor targets in patient treatment.

. Tyrosine Kinase PamChip® Array substrates.

Recognizing that tumor hypoxia is a common determinant of resistance to cytotoxic therapies and metastatic behavior, our prospective non-randomized study Locally Advanced Rectal Cancer – Radiation Response Prediction (LARC-RRP) of neoadjuvant chemotherapy and radiation followed by surgery and no further treatment in LARC offered a unique opportunity to explore this intriguing concept in a defined clinical context. Our hypothesis was that in LARC, with its extensive growth within the pelvic cavity, subclones with adaptation responses integrated by the PI3K signaling complex in particular [42] might expand under the selection pressure of hypoxic microenvironmental niches [29], [30]. The study was approved by the Institutional Review Board and the Regional Committee for Medical and Health Research Ethics of South-East Norway, and was in accordance with the Helsinki Declaration. Written informed consent was required for participation.

Utilizing the Tyrosine Kinase PamChip® Array technology to analyze patients’ tumor samples, the study might enable the identification of potentially actionable therapy targets implicated in hypoxic tumor signaling. The present report summarizes our findings presented in three previous communications [37], [38], [39] and further discusses opportunities and challenges of the kinase activity biomarker technology in light of the findings.

Section snippets

Study workup and treatment

The patient population reported here was enrolled from October 2005 through February 2008. Eligibility criteria included histologically confirmed rectal adenocarcinoma that was either T4 tumor, or T3 tumor or tumor of any T stage with lymph node involvement within 3 mm of the predicted circumferential resection margin, as assessed by magnetic resonance (MR) imaging, good performance status, and adequate organ function. Patient evaluation included rigid proctoscopy, pelvic examination by MR

LARC-RRP – patients and outcome

By May 2008, a study population of 67 patients was present for analysis of baseline tumor kinase activity [37]. For consistency in the present summary of data presented in the previously published reports [37], [38], [39], patients with synchronous resectable liver or lung metastases who were found eligible as per study protocol (n = 4), a patient who proceeded to palliative surgery following the neoadjuvant treatment (n = 1), and cases in which baseline tumor status was ambiguous (n = 4) are omitted

LARC-RRP – treatment outcome

This biomarker study collates the results from analysis of composite tumor kinase activities using the Tyrosine Kinase PamChip® Array technology in patients enrolled onto the LARC-RRP study. The estimated 5-year PFS (71%) was in accordance with recently reported data [4].

The described radiologic and histologic disease characteristics were also typical for LARC. Patients with T2–3 disease were more prone to achieve good response to the neoadjuvant treatment than those with organ-infiltrating

Conclusion

In order to optimize and individualize therapeutic efficacy of kinase-inhibitory drugs, it seems rational to exploit patients’ specific tumor kinase activities as functional biomarker of druggability. In taking advantage of dysregulated kinase signaling pathways as predictive biomarkers, the kinase substrate array technology is a rapid and high-throughput tool for biological profiling. Using such biomarker-finding technology in LARC, high tumor signaling integrated by the PI3K complex was

Conflict of interest statement

The authors declare no conflicts of interest.

Reviewers

Professor Hans-Joachim Schmoll, Martin-Luther-Universitat Halle-Wittenberg, Innere Med. IV, Ernst-Grube-Strasse 40, D-06120 Halle, Germany.

Dr. Kanwal P. Raghav, MD, MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, United States.

Acknowledgments

We greatly acknowledge the financial support by the European Union 7th Framework Programme (Grant 222741-METOXIA to A.H. Ree and K. Flatmark), South-Eastern Norway Regional Health Authority (Grants 2010014 and 2012002 to A.H. Ree), Norwegian Cancer Society (Grant 2006183 to A.H. Ree), and Akershus University Hospital (Grant 2010003 to A.H. Ree). The funding sources had no involvement in the study design, data collection and analysis, preparation of the manuscript, or decision to submit the

Professor Anne Hansen Ree is Consultant Clinical Oncologist at Akershus University Hospital in Lørenskog (Norway) and Professor of Clinical Oncology at University of Oslo in Oslo (Norway). She has been Principal Investigator of several translational therapy trials in advanced colorectal cancer.

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  • Cited by (0)

    Professor Anne Hansen Ree is Consultant Clinical Oncologist at Akershus University Hospital in Lørenskog (Norway) and Professor of Clinical Oncology at University of Oslo in Oslo (Norway). She has been Principal Investigator of several translational therapy trials in advanced colorectal cancer.

    Professor Kjersti Flatmark is Consultant Gastroenterological Surgeon at Oslo University Hospital in Oslo (Norway) and Professor of Surgery at University of Oslo. She has been Principal Investigator of several translational therapy trials in advanced colorectal cancer.

    Dr. Marie Grøn Sælen holds a PhD in molecular biology of tumor hypoxia in rectal cancer from University of Oslo. She is currently Resident in Oncology at University Hospital of North Norway in Tromsø (Norway).

    Dr. Sigurd Folkvord holds a PhD in molecular radiobiology in rectal cancer from University of Oslo. He is currently Resident in Surgery at Hedmark Central Hospital in Hamar (Norway).

    Dr. Svein Dueland is Consultant Clinical Oncologist at Oslo University Hospital and holds a PhD in cellular biology from University of Oslo. He has been Principal Investigator of numerous early-phase therapy trials in advanced cancer.

    Professor Jürgen Geisler is Consultant Clinical Oncologist at Akershus University Hospital and Professor of Clinical Oncology at University of Oslo. He has been Principal Investigator of numerous early-phase therapy trials in advanced cancer.

    Dr. Kathrine Røe Redalen holds a PhD in imaging physics and molecular radiobiology from University of Oslo. She is currently Postdoctoral Fellow and Principal Investigator of a translational biomarker trial in rectal cancer at Akershus University Hospital.

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