Expression of the scaffold connector enhancer of kinase suppressor of Ras 1 (CNKSR1) is correlated with clinical outcome in pancreatic cancer

While advances in the understanding of cancer biology, screening and risk-reducing interventions, and improved treatments have significantly reduced cancer mortality overall, pancreatic cancer remains a deadly disease. The American Cancer Society estimates 53,070 new cases and 41,780 deaths from pancreatic cancer in the United States during 2016 and predicts that pancreatic cancer will rank second of all cancer-related mortalities by the year 2030 [1, 2]. Neither current chemotherapy nor molecular therapy provides patients with an extension of survival measured by more than a few months, or the hope for sustained tumor regressions. Even in the minority of patients who are able to undergo surgical resection, median overall survival remains poor [3]. To date, only a few biomarkers have been associated with survival outcomes in pancreatic cancer [3, 4]. Considering the variability of clinical outcome and the uncertainty of the role of surgical resection in cancers at high risk for early progression, prognostic biomarkers accurately stratifying patients for individualized clinical decision-making would fill an unmet clinical need.

Activating somatic KRAS mutations are nearly omnipresent and a hallmark in the genetic make-up of pancreatic ductal adenocarcinoma (PDAC) [5]. While KRAS mutations themselves have been associated as prognostic markers, there is considerable and significant heterogeneity in the activation states of the downstream MAPK/ERK pathway, the molecular landscape, response to therapy, and clinical outcome across pancreatic cancers [6‐9]. The MAPK/ERK (MEK) pathway downstream of RAS has been the topic of significant research efforts in attempting to target and inhibit the oncogenic progression of RAS-mutant tumors. Certain scaffolding kinase proteins are essential to the spatiotemporal regulation of MAPK/ERK pathway signaling, as well as for regulating and integrating input from and output to other key signaling pathways involved in cellular homeostasis [10, 11]. The Kinase Suppressor of Ras-1 (KSR1) is a well-examined scaffolding protein; it has been shown to mediate tumor progression in pancreas cancer and may govern response to treatment [12, 13]. For example, KSR1 can compete with binding partners of BRAF and directly modulate response to small molecule inhibition of the MAPK pathway at the RAF level and it has been shown to be dysregulated in endometrial and colon cancers [14‐16]. CNKSR1 (connector enhancer of the Kinase Suppressor of Ras-1), a regulator and binding partner of KSR1, is another scaffolding protein which is less understood. Its role in pancreatic cancer biology, or as a biomarker, remains to be explored.

Current data suggests that CNKSR1 has multiple roles cancer biology, with some reports demonstrating that CNKSR1 interacts with tumor suppressors and others describe its scaffolding protein interactions as oncogenic [17, 18]. These include, for example, connecting Ephrin B stimulation via small GTPases to c-Jun N-terminal kinase (JNK) activation resulting in net cancer cell migration, or promoting cancer cell proliferation through the Akt-FoxO signalling axis [19, 20]. Recent studies using phosphomimetic mutants of CNKSR1 have identified phosphorylation sites in the scaffold critical for nuclear translocation and activation of MAPK pathway genes [21]. However, to date all CNKSR1 analysis in the context of pancreatic cancer has been performed at a molecular level with no translational or clinically oriented application. Using pancreatic tumor tissues from three independent cohorts, we aimed to evaluate the expression levels of CNKSR1 and its association with clinicopathological parameters and survival in pancreatic cancer. In addition we aimed to assess the association of CNKSR1 expression levels with MAPK pathway activity as measured by phospho-ERK. The observed association of CNKSR1 expression and survival outcome suggests scaffolding proteins of the RAS-MAPK pathway may account, in part, for the observed heterogeneity of PDAC biology, and clinically may aid in improved future patient stratification.

In this study, we examined the expression levels of the scaffolding kinase CNKSR1 in pancreatic cancer surgical resection specimens and evaluated their impact on clinical outcome, including overall survival (OS). We found, in a limited set of matched normal and tumor specimens, that CNKSR1 expression is elevated in cancer tissue compared to normal uninvolved pancreas tissue. Elevated CNKSR1 expression correlated with improved survival as an independent prognostic marker using multivariate analysis, with patients in the low CNKSR1 expression group having a median OS that is nearly half that of patients with high CNKSR1 expression. In addition, we attempted to determine whether CNKSR1 status might affect the survival difference associated with resection in pancreatic cancer patients. If validated in a larger patient sample, such information might be useful in pre-operative decision-making.

CNKSR1 expression has not been previously shown to be associated with survival. This is in contrast to KSR1, the other major scaffolding protein of the MAPK/ERK pathway. KSR1, which tightly regulates MAPK/ERK signaling output, has been shown to be overexpressed in endometrial and colon carcinoma and high expression levels reported to be associated with decreased survival in breast cancer [14, 15, 27]. KSR1 has been shown to impact tumor biology, including in preclinical models of pancreas cancer, as well as response to chemotherapy [12]. To date, such information is largely lacking for CNKSR1, a binding partner and regulator of KSR1 and the MAPK pathway.

One of the main functions of the scaffold protein CNKSR1 is the regulation of other cancer-related signaling pathways integrating output to different intracellular signaling cascades upon cellular stimulation by extracellular cues [11, 12, 17‐20]. In breast and cervical cancer cell models, a pro-oncogenic potential has been demonstrated through ERK-independent AKT-FoxO and NF-kappaB pathways [17, 19]. In a colorectal cancer cell model, CNKSR1 has been shown to facilitate pro-apoptotic signaling via RASSF1A [18]. Importantly, a recent cell-based study expressing mutants of known phosphorylation sites of CNKSR1 in cancer cells identified sites linked to nuclear translocation with concomitant activation of MAPK pathway-driven serum response element (SRE) gene expression providing, for the first time, a direct correlation of cellular CNKSR1 distribution and MAPK pathway signaling output. [21] These findings are in line with the identified correlation of CNKSR1 cellular distribution (cytoplasmic only vs cytoplasmic and nuclear) and nuclear p-ERK expression levels observed in our study. The finding that pancreatic tumors which have nuclear CNKSR1 expression in addition to the predominant cytoplasmic expression of CNKSR1 show higher nuclear p-ERK expression levels suggests that cellular localization, rather than absolute CNKSR1 expression levels, might be one of the mechanisms of CNKRS1-mediated control of MAPK pathway activity. In this regard, Fisher and colleagues showed in an elegant study with a set of different phosphomimetic mutant constructs that phosphorylation of Tyr 519 recruits CNKSR1 to the nucleus and phosphorylation of Tyr 26 enables CNKSR1 to activate MAPK pathway-governed serum response element (SRE) gene transcription [21]. Previous smaller correlative biomarker studies did show that increased nuclear p-ERK staining levels were associated with poorer survival in pancreas cancer [28, 29]. Such MAPK pathway regulation by nuclear translocation possibly induced by posttranslational modifications including phosphorylation of select sites of CNKSR1, appears in line with our findings of a correlation between the cellular distribution of CNKSR1 and nuclear p-ERK expression levels [30].

While there have been more than 100 correlative biomarkers described investigating pancreatic cancer, there have been less than a dozen studies which correlated putative markers with survival outcome [31]. Similar to the hazard ratio (HR) estimates for death reported for CNKSR1 in this study, the majority of previously described prognostic biomarker studies reported HRs for death ranging from 1.5 to 4 [31]. Exceptions to these studies are, among others, the recently reported study of 13 putative pancreas cancer biomarkers from investigators at the Memorial Sloan-Kettering Cancer Center (MSKCC) describing two biomarkers with HRs exceeding ten [4]. It is also noteworthy that of the biomarkers reported to be associated with survival (reviewed by Winter et al. 2013), there are a number of proteins either indirectly or directly connected to MAPK/ERK signaling (Ras, ERBB2, Myc) [4, 31].

A limitation of this study was that the dataset did not include all clinical variables, such as complete TNM staging and chemotherapy treatment, and thus we could not evaluate all variables that might impact survival. While the HRs of known clinical (TNM system N and M stage) and pathological variables in the Cox proportional hazard ratio model yielded previously known associations with outcome, the T variable of the AJCC TNM staging system was not fully collected and only incompletely captured by the stage variable (localized vs regional). While this somehow limits direct comparability to previous studies, prospectively evaluated and validated nomograms on surgical outcome of pancreas cancer have shown that, by far, M and N stage, as captured in our study, are the strongest predictors of outcome [32, 33]. In addition, data regarding systemic therapy was not available, though nearly all cases were diagnosed prior to the routine use of FOLFIRINOX or Gemcitabine/nab-Paclitaxel.

We consider validation of the CNKSR1 expression pattern used for the correlative outcome studies in the two independent cohorts of pancreas cancer surgical specimens a strength of the study. Co-staining of matched SEER Pancreatic Cancer TMA specimens with p-ERK suggests a correlation between cellular distribution of CNKSR1 and nuclear p-ERK expression levels, possibly indicating regulation of MAPK pathway signaling less connected to absolute CNKSR1 expression levels. In addition, the SEER TMA studied has been used in multiple previous biomarker studies, several of which have been validated in additional larger cohorts [4, 22]. CNKSR1 may represent an additional prognostic marker in pancreatic cancer, and further validation in a larger cohort of recent patients is needed.

In this correlative tissue study including independent pancreatic cancer tissue microarrays from different sources, CNKSR1 expression was found to be an independent marker of patient outcome. We identified CNKSR1 low patients at high risk for disease progression suggesting that there might be distinct differences in tumor biology between CNKSR1 high and CNKRS1 low tumors. In CNKSR1 low expressing tumors, patients did not show any difference in survival when stratified by resection status whereas patients with high CNKSR1 expression levels who underwent resection had significantly improved outcome compared to non-resected patients in this group. Combination of CNKSR1 expression levels with current clinicopathological prognostic features might improve risk stratification and treatment selection.