Phosphorylation Of p27Kip1 Research Articles

Abstract 458: Targeting p27Kip1 to inhibit CDK4/6/2 as a novel therapeutic strategy to treat pancreatic ductal adenocarcinoma

Abstract CDK4/6 inhibitors are powerful tools used clinically to treat metastatic, HR+ BC patients. While combined CDK4/6i treatment in combination with Estrogen modulation significantly extends Progression Free Survival (PFS) in metastatic HR+ breast cancer patients, those treated invariably develop aquired resistance, which results in only marginal improvement in Overall Survival (OS). The drug resistance seen in the presence of CDK4/6i, such as palbociclib, has been shown to be attributed to activation and compensation by CDK2. Pancreatic ductal adenocarcinoma (PDAC) is characterized by aberrant activation of the CyclinD-CDK4/6 signaling pathway and inactivation of the CDKN2A locus encoding the CDK4/6 inhibitor, p16INK4A, and was initially thought to be a good candidate for CDK4/6i use. Surprisingly, CDK4/6i do not show response in most PDAC lines and have not been used clinically in this tumor type. We hypothesized that PDAC may be intrinsically resistant to CDK4/6 inhibition due to activation of CDK2 and as such inhibition of both CDK4/6 and CDK2 may be required. We have developed a novel strategy to kill drug-resistant tumor cells using a therapeutic liposomal:peptide formulation, IpY.1, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. IpY.1 is specific for p27, and this reduces toxicity relative to that seen with the small molecule ATP-competitive CDK4/6i or CDK2i. IpY.1 blocks DNA replication in HR+ breast cancer (BC) cells and mouse models, but unlike CDK4/6i or CDK2i treatment, it induces ROS-dependent necroptosis to kill the tumor cells. Palbociclib did not have a significative effect on ATP levels or proliferation of PDAC cell lines. In contrast, we found that IpY.1 was able to block proliferation and reduce viability in palbociclib-resistant PDAC lines, suggesting that dual CDK4/6 and CDK2 inhibition via p27 targeting was required. Some PDAC lines were “super sensitive” and responded with IC50 values ~10X less than seen with the ER+ BC lines, and we are currently screening for biomarkers of response. CDK2 activity was reduced in IpY.1 treated lines and markers of necroptosis were detected. Immunoblot analysis showed decreased pY88 phosphorylation of p27 (the target of IpY) demonstrating target engagement. We have demonstrated that unlike palbociclib, IpY.1 blocks proliferation and viability in all PDAC lines tested, suggesting that targeting p27 may be a viable approach to treat PDAC. Citation Format: Carolina Guido, Susan RS Gottesman, Stacy W. Blain. Targeting p27Kip1 to inhibit CDK4/6/2 as a novel therapeutic strategy to treat pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 458.

Abstract 1860: NP-ALT, a liposomal:peptide drug, blocks p27Kip1 phosphorylation to cause cell death in CDK4i-resistant breast cancer

Abstract Resistance to cyclin D-cdk4/6 inhibitors (CDK4/6i) is frequently caused by compensatory CDK2 activity. We have developed a novel strategy to kill CDK4i resistant tumor cells using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. IpY is specific for p27, and this reduces toxicity relative to that seen with the small molecule ATP-competitive CDK4/6i or CDK2i. NP-ALT blocks DNA replication in HR+ breast cancer (BC) cells and mouse models, but unlike CDK4/6i or CDK2i treatment, it induces ROS-dependent necroptosis to kill the tumor cells. This RIPK1/RIPK3-dependent cell death is more specific for HR+ lineages, but the combination of NP-ALT and palbociclib in TNBC cells causes synergy to induce necroptosis. We found that NP-ALT was still able to induce necroptosis in cell lines resistant to palbociclib, including those harboring genetic perturbations detected clinically in patients resistant to palbociclib. HR+ cell lines engineered to overexpress CDK6, FGFR, cyclin E, or cyclin D or ones that have loss of FAT1 or RB were still sensitive to NP-ALT. We tested other CDK2i, including CYC065, a CDK2/9 inhibitor and PF-06873600, a CDK4/6/2 inhibitor, and and found that while they were able to block DNA replication, only NP-ALT was able to induce ROS-dependent death in the resistant lines. We have shown that IpY reduces tumor volumes in resistant in vivo models as well, without causing adverse effects. Thus, NP-ALT and p27 targeting has several differentiators, which might provide clinical advantages: a) its low toxicity due to the tight specificity of the peptide for the p27 target and b) its ability to kill tumor cells as opposed to just causing cytostasis. Concarlo is currently developing NP-ALT into a therapeutic lead for the treatment of drug-resistant breast cancer. Citation Format: Stacy Wister Blain, Arianna Chand, Lina Pedraza-Ortiz, Allison VanInwegen, Lingyu Yan, Yun Wu, Grace Chen, Irina Jilishitz. NP-ALT, a liposomal:peptide drug, blocks p27Kip1 phosphorylation to cause cell death in CDK4i-resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1860.

P27kip1 expression and phosphorylation dictate Palbociclib sensitivity in KRAS-mutated colorectal cancer

In colorectal cancer, mutation of KRAS (RASMUT) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RASMUT colorectal cancer cells, we observed an increased expression of p27kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27kip1 showed variable expression in RASMUT colorectal cancer samples, our study supports the possibility that p27kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.

NP-ALT, a Liposomal:Peptide Drug, Blocks p27Kip1 Phosphorylation to Induce Oxidative Stress, Necroptosis, and Regression in Therapy-Resistant Breast Cancer Cells.

Resistance to cyclin D-CDK4/6 inhibitors (CDK4/6i) represents an unmet clinical need and is frequently caused by compensatory CDK2 activity. Here we describe a novel strategy to prevent CDK4i resistance by using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. We find that NP-ALT blocks proliferation in HR+ breast cancer cells, as well as CDK4i-resistant cell types, including triple negative breast cancer (TNBC). The peptide ALT is not as stable in primary mammary epithelium, suggesting that NP-ALT has little effect in nontumor tissues. In HR+ breast cancer cells specifically, NP-ALT treatment induces ROS and RIPK1-dependent necroptosis. Estrogen signaling and ERα appear required. Significantly, NP-ALT induces necroptosis in MCF7 ESRY537S cells, which contain an ER gain of function mutation frequently detected in metastatic patients, which renders them resistant to endocrine therapy. Here we show that NP-ALT causes necroptosis and tumor regression in treatment naïve, palbociclib-resistant, and endocrine-resistant BC cells and xenograft models, demonstrating that p27 is a viable therapeutic target to combat drug resistance. IMPLICATIONS: This study reveals that blocking p27 tyrosine phosphorylation inhibits CDK4 and CDK2 activity and induces ROS-dependent necroptosis, suggesting a novel therapeutic option for endocrine and CDK4 inhibitor-resistant HR+ tumors.

Abstract LB115: Blocking p27Kip1phosphorylation with a liposomal:peptide drug induces Reactive Oxygen Species,necroptosis and tumor regression in breast cancer cells

Abstract Resistance to cyclin D-cdk4/6 inhibitors (CDK4/6i) represents an unmet clinical need and is frequently caused by compensatory CDK2 activity. Here we describe a novel strategy to prevent CDK4i resistance by using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. We find that NP-ALT blocks proliferation in HR+ breast cancer (BC) cells, as well as CDK4i-resistant cell types, including Triple Negative (TN) BC. The peptide ALT is not as stable in primary mammary epithelium, suggesting that NP-ALT has little effect in non-tumor tissues. In HR+ BC cells specifically, NP-ALT treatment induces ROS and RIPK1-dependent necroptosis. Estrogen signaling and ERα appear required. Significantly, NP-ALT induces necroptosis in MCR7 ESRY537S cells, which contain an ER gain of function mutation frequently detected in metastatic patients, which renders them resistant to endocrine therapy. Here we show that NP-ALT causes necroptosis and tumor regression in treatment naïve, palbociclib-resistant and endocrine-resistant BC cells and xenograft models, demonstrating that p27 is a viable therapeutic target to combat drug resistance. Because the RAS/MAPK axis is the target of many therapies, NP-ALT, with its ability to target p27, can deal with the backend drug resistance seen in the presence of other inhibitors, extending the addressable market of this line of therapy. Citation Format: Stacy Wister Blain, Irina Jilishitz, Jason L. Quinones, Priyank Patel, Grace Chen, Jared Pavetsky, Allison VanInwegen VanInwegen, Scott Schoninger, Manasi P. Jogalekar, Vladislav Tsiperson, Lingyue Yan, Yun Wu, Susan R. Gottesman, Jonathan Somma. Blocking p27Kip1phosphorylation with a liposomal:peptide drug induces Reactive Oxygen Species,necroptosis and tumor regression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB115.

Characterization of Signalling Pathways That Link Apoptosis and Autophagy to Cell Death Induced by Estrone Analogues Which Reversibly Depolymerize Microtubules

The search for novel anti-cancer compounds which can circumvent chemotherapeutic drug resistance and limit systemic toxicity remains a priority. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10)15-tetraene-3-ol-17one (ESE-15-one) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16) are sulphamoylated 2-methoxyestradiol (2-ME) analogues designed by our research team. Although their cytotoxicity has been demonstrated in vitro, the temporal and mechanistic responses of the initiated intracellular events are yet to be determined. In order to do so, assays investigating the compounds’ effects on microtubules, cell cycle progression, signalling cascades, autophagy and apoptosis were conducted using HeLa cervical- and MDA-MB-231 metastatic breast cancer cells. Both compounds reversibly disrupted microtubule dynamics as an early event by binding to the microtubule colchicine site, which blocked progression through the cell cycle at the G1/S- and G2/M transitions. This was supported by increased pRB and p27Kip1 phosphorylation. Induction of apoptosis with time-dependent signalling involving the p-JNK, Erk1/2 and Akt/mTOR pathways and loss of mitochondrial membrane potential was demonstrated. Inhibition of autophagy attenuated the apoptotic response. In conclusion, the 2-ME analogues induced a time-dependent cross-talk between cell cycle checkpoints, apoptotic signalling and autophagic processes, with an increased reactive oxygen species formation and perturbated microtubule functioning appearing to connect the processes. Subtle differences in the responses were observed between the two compounds and the different cell lines.

WIP1 dephosphorylation of p27Kip1 Serine 140 destabilizes p27Kip1 and reverses anti-proliferative effects of ATM phosphorylation

ABSTRACT The phosphoinositide-3-kinase like kinases (PIKK) such as ATM and ATR play a key role in initiating the cellular DNA damage response (DDR). One key ATM target is the cyclin-dependent kinase inhibitor p27Kip1 that promotes G1 arrest. ATM activates p27Kip1-induced arrest in part through phosphorylation of p27Kip1 at Serine 140. Here we show that this site is dephosphorylated by the type 2C serine/threonine phosphatase, WIP1 (Wildtype p53-Induced Phosphatase-1), encoded by the PPM1D gene. WIP1 has been shown to dephosphorylate numerous ATM target sites in DDR proteins, and its overexpression and/or mutation has often been associated with oncogenesis. We demonstrate that wildtype, but not phosphatase-dead WIP1, efficiently dephosphorylates p27Kip1 Ser140 both in vitro and in cells and that this dephosphorylation is sensitive to the WIP1-specific inhibitor GSK 2830371. Increased expression of wildtype WIP1 reduces stability of p27Kip1 while increased expression of similar amounts of phosphatase-dead WIP1 has no effect on p27Kip1 protein stability. Overexpression of wildtype p27Kip1 reduces cell proliferation and colony forming capability relative to the S140A (constitutively non-phosphorylated) form of p27. Thus, WIP1 plays a significant role in homeostatic modulation of p27Kip1 activity following activation by ATM.

Abstract LB-224: Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer

Abstract PURPOSE: Cdk4 inhibitors (CDK4i), such as palbociclib, are approved in combination with hormonal therapy as a front line treatment for metastatic HR+, Her2- breast cancer. However, despite these advances, many patients demonstrate either de novo or acquired resistance. This highlights the need to identify biomarkers to pinpoint patients who would respond to this therapy. In Rb+ tumors, which comprise the majority of HR+ breast cancers, Ki67, Cyclin D, cdk4, and p16 levels do not identify responsive subgroups. Cyclin D or cdk4 levels themselves are not reliable because tyrosine phosphorylated p27Kip1 (pY88) is required for activation of the cyclin D-cdk4 (DK4) complex. We have previously shown that the level of pY88 correlates with cdk4 activity: the more pY88 detected, the greater the specific activity of cdk4 (Patel, et. al. MCR, 2018). We developed dual immunohistochemical staining for p27 and pY88 and demonstrated that pY88 status, which is negative in benign breast epithelium, stratified tumors across all hormone receptor groups (Gottesman, et.al, MCR, 2018). Lack of pY88 suggested that DK4 was inactive, and that these samples would not have the active cdk4 target. We have grown tumor resection material in explant culture, treated with palbociclib, and stained with Ki67 as a marker of response. Explants from the no pY88 group were non-responsive, while explants from the low or high pY88 group responded to drug. Thus, we hypothesized the pY88-p27 status may serve as a biomarker for patients that can respond to cdk4i therapy. RESULTS: We analyzed paraffin-embedded archival breast cancer biopsies from a 13 patient cohort of HR+, Her2- patients who had received Palbociclib/Letrozole in the front line metastatic setting. 62% of these patients progressed within 8 months of treatment, while 38% were on drug for 12-38 months. Using our staining method, we stratified patients into three groups based on pY88 status: 23% had no staining (Group 0), 38% had a low percentage of pY88+ cells (Group 1), and 38% had a high percentage of pY88+ cells (Group 2, >30% of cells pY88+). We then compared the time until disease progression, when they were removed from drug, for each group. Patients with a pY88 score of 0 had disease progression within 6-8 months, suggesting that they exhibited primary resistance. Patients with a pY88 score of 2 also showed disease progression within 6 months. This suggested that the Y88 high group may have too much active cdk4 target to respond to pharmacological drug dose, a hypothesis supported by our breast cancer cell line data. However, patients with a low pY88 score were all still on drug after 12+ months with either reduced tumor or stable disease. Additional patients continue to be recruited. CONCLUSION: Our data suggest that pY88-p27 status, as a surrogate marker for cdk4 activity, associates with responsiveness to CDK4i treatment. Clinical use of the pY88 biomarker may identify patients responsive or resistant to Cdk4 targeting drugs. Citation Format: Stacy W. Blain, Susan R.S. Gottesman, Jonathan Somma, Vladislav Tsiperson, Julia McGuinness, Matthew Ingham, Kevin Kalinsky, Gary Schwartz, Elina Tress, Hanina Hibshoosh, Mohamed Mh Kahila, Bachar Samra Samra, Evelyn Taiwo, Steve Xie. Tyrosine phosphorylation of p27Kip1 associates with Palbociclib responsiveness in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-224.

Tyrosine Phosphorylation of p27Kip1 Correlates with Palbociclib Responsiveness in Breast Cancer Tumor Cells Grown in Explant Culture.

Cdk4-targeting drugs, such as palbociclib, are approved for metastatic ER/PR+, Her2- breast cancer. However, other than loss of retinoblastoma, which is very rare in this subset, there are no biomarkers to predict response. Cyclin D or cdk4 levels are not by themselves indicative, because p27Kip1 is required for cyclin D-cdk4 complex activation. Tyrosine phosphorylation of p27, including modification on residue Y88 (pY88), activates DK4-p27, and the pY88 level correlates with palbociclib responsiveness in cell lines. We developed dual IHC staining for p27 and pY88, and found that benign breast epithelium was negative, while breast cancer biopsies (of varied hormonal status) could be stratified for pY88 status. Lack of pY88 suggested that DK4 was inactive, and that these samples would not have the target required for palbociclib response. Tumor resection material was grown in explant culture, treated with palbociclib, and stained with Ki67 as a marker of response. Explants from the no pY88 group were nonresponsive, while explants from the low or high pY88 group responded to drug. IMPLICATIONS: Use of the pY88 biomarker, as a surrogate for cdk4 activity, may identify patients responsive to cdk4-targeting drugs and expand use of this therapy.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/3/669/F1.large.jpg.

CACYBP Enhances Cytoplasmic Retention of P27Kip1 to Promote Hepatocellular Carcinoma Progression in the Absence of RNF41 Mediated Degradation.

Calcyclin-binding protein (CACYBP) is a multi-ligand protein implicated in the progression of various human cancers. However, its function in hepatocellular carcinoma (HCC) remains unknown.Methods: The expression of CACYBP and RNF41 (RING finger protein 41) in HCC cancer and adjacent non-tumor tissues was detected by immunohistochemistry. CCK-8 assays, colony formation assays, flow cytometry detection and xenograft models were used to evaluate the impact of CACYBP expression on HCC cell growth, apoptosis and cell cycle regulation. Immunoprecipitation and ubiquitination assays were performed to determine how RNF41 regulates CACYBP. The regulatory mechanism of RNF41-CACYBP signaling axis on P27Kip1 was investigated by western blotting and immunofluorescence.Results: CACYBP was highly expressed and associated with poor prognosis in HCC. CACYBP expression was required for HCC cell growth in vitro and in vivo. Moreover, we identified RNF41 as a specific binding partner of CACYBP at exogenous and endogenous levels. RNF41 recruited CACYBP by its C-terminal substrate binding domain, subsequently ubiquitinating CACYBP and promoting its degradation in both proteasome- and lysosome-dependent pathways. In HCC tissues, RNF41 expression was reduced and conferred a negative correlation with CACYBP expression. Mechanistically, CACYBP overexpression stimulated the Ser10, Thr157 and Thr198 phosphorylation of P27Kip1 and its cytoplasmic retention, and RNF41 co-expression attenuated this phenomenon. CACYBP depletion led to decreased levels of cyclin D1, cyclin A2, CDK2 and CDK4, causing a typical cell cycle arrest at G1/S phase and increasing apoptosis in HCC cells. P27Kip1-S10D but not P27Kip1-S10A reconstitution rescued partially the cell cycle function and apoptotic feature after CACYBP depletion.Conclusion: Our findings provide novel insights into the functional role and regulatory mechanism of CACYBP in HCC.

The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells.

SummarySkeletal muscle stem cell (MuSC) function declines with age and contributes to impaired muscle regeneration in older individuals. Acting through AMPK/p27Kip1, we have identified a pathway regulating the balance between autophagy, apoptosis, and senescence in aged MuSCs. While p27Kip1 is implicated in MuSC aging, its precise role and molecular mechanism have not been elucidated. Age-related MuSC dysfunction was associated with reduced autophagy, increased apoptosis, and hypophosphorylation of AMPK and its downstream target p27Kip1. AMPK activation or ectopic expression of a phosphomimetic p27Kip1 mutant was sufficient to suppress in vitro apoptosis, increase proliferation, and improve in vivo transplantation efficiency of aged MuSCs. Moreover, activation of the AMPK/p27Kip1 pathway reduced markers of cell senescence in aged cells, which was, in part, dependent on p27Kip1 phosphorylation. Thus, the AMPK/p27Kip1 pathway likely regulates the autophagy/apoptosis balance in aged MuSCs and may be a potential target for improving muscle regeneration in older individuals.

Abstract 2309: Increase in Cdk4 activity during progression of small intestinal neuroedocrine tumors

Abstract In the USA, there are approximately 5000 new cases of small intestinal neuroendocrine tumors (SINETs) per year, many of which already show liver metastasis upon initial diagnosis. The genetic causes of SINETs have long been a mystery. The only known recurrent mutations are in the Cdkn1b gene, which is mutated in 8% of tumors. Cdkn1b encodes p27kip1, an inhibitor of Cdk4 and Cdk2. We analyzed proteins and genes linked to the function of Cdk4 in a set of early and late SINETs (pathological stages M0 or M1). Amplification of Cdk4 activators (CcnD1, CcnD2, CcnD3), or loss of copy of Cdk4 inhibitors (Cdkn1b as well as Cdkn2a), or mutation of Cdkn1b were uncommon events, but notably occurred almost exclusively in patients with late stage disease. Patients with late stage tumors also had genetic abnormalities in Phlda3, including loss of copy, loss of heterozygosity, or presence of the rs35383942 SNP. Altogether, more than 50 percent of the advanced tumors had one or more of these genetic events, compared to less than 15 percent of the early tumors. Phlda3 is known to affect Akt activity which in turn is known to repress p27kip1 by phosphorylation, and indeed we detected more p27kip1 phosphorylation in late stage tumors. Finally, advanced tumors showed higher expression of Cdk4 and increased phosphorylation of serine-807 or serine-811 of the Rb1 protein. Together, these data argue that there is genetic pressure to increase Cdk4 activity during progression of SINETs, and suggest that patients with advanced SINETs may respond to Cdk4 inhibitors. Citation Format: Tanupriya Contractor, Chris R. Harris. Increase in Cdk4 activity during progression of small intestinal neuroedocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2309.

Dual Inhibition of CDK4 and CDK2 via Targeting p27 Tyrosine Phosphorylation Induces a Potent and Durable Response in Breast Cancer Cells.

Cyclin-dependent kinase 4/6 (CDK4/6)-specific inhibitors, such as palbociclib, have shown clinical efficacy, but primary or secondary resistance has emerged as a problem. To develop more effective therapeutic approaches, investigation is needed into the mechanisms of resistance or adaption. Here, it is demonstrated that CDK2 compensates for loss of CDK4 activity to rescue palbociclib-arrested breast cancer cells, suggesting that inhibition of both kinases is required to achieve durable response. In addition, a novel strategy is described to inhibit tyrosine phosphorylation of p27Kip1 (CDKN1B) and simultaneously inhibit both CDK2 and CDK4. p27Kip1 is a required assembly factor for cyclin-CDK4 complexes, but it must be phosphorylated on residue Y88 to open or activate the complex. The Brk-SH3 peptide, ALT, blocks p27 Y88 phosphorylation, inhibiting CDK4. Nonphosphorylated p27 is no longer a target for ubiquitin-mediated degradation and this stabilized p27 now also inhibits CDK2 activity. Thus, ALT induction inhibits both the kinase that drives proliferation (CDK4) and the kinase that mediates resistance (CDK2), causing a potent and long-lasting cell-cycle arrest. ALT arrests growth of all breast cancer subgroups and synergizes with palbociclib to increase cellular senescence and to cause tumor regression in breast cancer xenograft models. The use of ALT demonstrates that both CDK4 and CDK2 need to be inhibited if long-term efficacy is to be achieved and represents a novel modality to inhibit breast cancer cells.Implications: Modulating tyrosine phosphorylation of p27 impacts both proliferative (CDK4) and resistance (CDK2) mechanisms in breast cancer and suggests that phospho-p27 status may serve as a biomarker for patients that are responsive to CDK4/6 inhibition. Mol Cancer Res; 16(3); 361-77. ©2018 AACR.

Chromosome region maintenance-1 (CRM1) regulates apoptosis of intestinal epithelial cells via p27kip1 in Crohn's disease

To investigate the role of chromosome region maintenance-1 (CRM1) in Crohn's disease (CD) and its potential pathological mechanisms. The expression and distribution of CRM1 in mucosal biopsies from patients with active CD and normal controls were detected by immunohistochemistry (IHC). We established a murine model of acute colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Western blot was performed to investigate the expression levels of CRM1, apoptotic markers (active caspase-3 and cleaved PARP), p27kip1 and p-p27ser10. IHC was performed to evaluate the distribution of CRM1, and double immunofluorescence (IF) was performed to evaluate the co-localization of CRM1 and active capase-3. Cells of the human intestinal epithelial cell line HT-29 were incubated with tumor necrosis factor-α (TNF-α) to establish an apoptotic in vitro model. Western blot was performed to determine the expression levels of CRM1, active caspase-3, cleaved PARP and p-p27ser10. Cytoplasmic and nuclear extracts were assessed to examine the translocation of CRM1. The interaction between CRM1 and p27kip1 was assessed by co-immunoprecipitation (co-IP) assays. Furthermore, we used small interfering RNA (siRNA) to knock down the protein expression of CRM1 in HT-29 cells and then measured the expression of active caspase-3, cleaved PARP and p-p27ser10. Flow cytometry was used to determine the effect of CRM1 on intestinal epithelial cell (IEC) apoptosis. We observed up-regulation of CRM1 accompanied by elevated levels of IEC apoptotic markers (active caspase-3 and cleaved PARP) and p-p27ser10 in IECs of patients with active CD and in TNBS-induced colitis model cells. However, the expression of p27kip1 was negatively correlated with the expression patterns of CRM1, p-p27ser10 and apoptotic biochemical markers. Co-localization of CRM1 and active caspase-3 in IECs of the TNBS group further indicated the possible involvement of CRM1 in IEC apoptosis. By employing TNF-α-treated HT-29 cells as an in vitro IEC apoptosis model, we found that the expression levels of CRM1 and p-p27ser10 were in accordance with active caspase-3 and cleaved PARP. In addition, immunoprecipitation confirmed the physical interaction between CRM1 and p27kip1. siRNA knockdown of CRM1 significantly inhibited the phosphorylation of p27kip1 and the expression of active caspase-3 and cleaved PARP. In addition, flow cytometry analysis also showed that silencing CRM1 by siRNA inhibited TNF-α-induced cellular apoptosis in HT-29 cells. Up-regulated CRM1 may facilitate IEC apoptosis possibly through p27kip1 in CD, indicating an important role of CRM1 in the pathophysiology of CD.

MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

BackgroundOverexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.Methods and ResultssiRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.ConclusionsMARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

P27Kip1 signaling: Transcriptional and post-translational regulation

p27Kip1 is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27Kip1 allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27Kip1 protein levels, in particular nuclear expressed p27Kip1, is implicated in both disease progression and poor prognosis in a variety of cancers. p27Kip1 expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. However, regulation of p27Kip1 protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27Kip1 at Thr187 and Ser10 is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser10 phosphorylated p27Kip1 is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27Kip1 in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27Kip1 localization and/or block its degradation.

Abstract POSTER-BIOL-1333: TRIP6 regulates p27KIP1 to promote ovarian tumorigenesis

Abstract Although the nuclear p27KIP1 functions as a negative regulator of G1/S cell cycle progression by binding to and inhibiting cyclin-CDK complexes, the cytosolic p27KIP1 has been shown to promote adhesion disassembly and tumor invasion. Loss of nuclear p27KIP1 and cytosolic mislocalization of p27KIP1 are frequently found during cancer progression, and these events correlate with poor clinical outcome in many types of cancers, including ovarian cancer. However, the mechanisms underlying this dysregulation are not yet fully understood. TRIP6 is an adaptor protein that is highly expressed in ovarian serous cystadenocarcinoma. It serves as a platform to recruit a number of molecules, such as NF-κB p65, c-Src, p130cas, Crk, LPA2 and Fas/CD95 receptors, to promote cell motility and antiapoptotic signaling. Using an ovarian cancer xenograft mouse model, we find that TRIP6 knockdown significantly reduces ovarian tumor proliferation. We determine that this effect is in part through the regulation of p27KIP1. Our data show that TRIP6 serves as a bridge to promote the recruitment of p27KIP1 to AKT in the cytosol. TRIP6 regulates the membrane translocation and activation of AKT, and facilitates AKT-mediated recognition and phosphorylation of p27KIP1 specifically at T157, thereby promoting the cytosolic mislocalization of p27KIP1. This is required for p27KIP1 to enhance LPA-induced ovarian cancer cell migration. TRIP6 also promotes serum-induced reduction of nuclear p27KIP1 expression through Skp2-dependent and -independent mechanisms. Consequently, knockdown of TRIP6 in ovarian cancer xenografts restores nuclear p27KIP1 expression and impairs tumor proliferation. Since the expression of TRIP6 and the activity of AKT are significantly higher in ovarian cancer, restoring the expression of nuclear p27KIP1 and inhibiting the migratory effect of phospho-T157-p27KIP1 by targeting TRIP6 may prove to be an effective approach to reduce ovarian cancer progression. Citation Format: Fang-Tsyr Lin, Victor T. G. Lin, Vivian Y. Lin, Yun-Ju Lai, Chen-Shan Chen, Kang Liu, Weei-Chin Lin. TRIP6 regulates p27KIP1 to promote ovarian tumorigenesis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1333.

Abstract P5-08-01: Tyrosine phosphorylation of p27kip1 regulates the activity of cyclin D-cdk4 complexes in breast cancer

Abstract The oncogenes Cyclin D and cdk4 are overexpressed in breast cancer, but the levels of these proteins are not always accurate indicators of oncogenic activity because p27Kip1 is required to assemble this otherwise unstable dimer. However, p27’s association activates or alternatively inhibits cyclin D-cdk4, serving as a bona fide ON/OFF "switch." Tyrosine (Y) phosphorylation of residues Y88/89 in p27 displaces its C-terminus from the cdk4 active site, permitting both ATP binding and CAK phosphorylation of cdk4’s T loop. This model leads to the following hypothesis: modulation of p27 pY controls cdk4 activity, which in turn regulates efficient cell cycle passsage, and in breast cancer where cdk4 activity is deregulated, p27 may be constitutively switched ON. Deregulated Src Family Kinase (SFK) signaling in cancer may increase p27 pY, constitutively activating oncogenic cdk4, causing continuous cell cycling. Using our p27 pY phosphospecific antibody, we have shown in primary tumors, that p27 pY is not detected in benign tissue regions, but is detected in grade 1 and progressively higher grade tumors, suggesting that p27 pY may be a marker for increased oncogenic cdk4 activity and cdk4 inhibitor sensitivity. We identified an SH3 recruitment domain within p27 that controls p27 pY, and in turn controls cdk4 activity. Blocking the SH3:p27 interaction with small peptides prevents p27 pY and cdk4 activity in vitro and in vivo. Using a phage-ELISA assay, we identified PTK6/Brk (Protein Tyrosine Kinase 6/Breast Tumor Kinase) that functions as a high-affinity kinase, able to phosphorylate p27 in vitro and associate with phosphorylated p27 in vivo. Overexpression of PTK6 in vivo increases p27 pY and increases resistance to specific cdk4 inhibition by the chemical inhibitor, PD0332991. An ALTernatively spliced form of PTK6 (ALT), which contains the SH3 domain, specifically associates with p27 in cells arrested by contact or serum-starvation, blocking pY and acting as an endogenous inhibitor of cdk4. As PTK6/Brk is overexpressed in more than 60% of human breast carcinomas, our data suggest that PTK6/Brk overexpression facilitates cell cycle progression by increasing cdk4 activity through direct p27 Y phosphorylation. As PD0332991 moves into the clinic, p27 pY could serve as a marker to identify tumors sensitive to cdk4 inhibition, while blocking the PTK6:p27 interaction with small molecules represents a novel therapeutic option to inhibit cdk4 activation. Citation Format: Priyank Patel, Elina Shetyn, Cindy Gomez, Susan RS Gottesman, Angela Tyner, Benedikt Asbach, Ralf Wagner, Stacy W Blain. Tyrosine phosphorylation of p27kip1 regulates the activity of cyclin D-cdk4 complexes in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-08-01.

Abstract 1325: Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells

Abstract Profilin-1 is a ubiquitously expressed actin-binding protein that is downregulated in human breast cancer. Overexpression of profilin-1 in human breast cancer cells suppresses their tumorigenic potential. We had previously shown that profilin-1 overexpression elevates the expression level of p27kip1 leading to cell-cycle arrest in G1 phase. In the present work, we explore the underlying mechanism. Our studies showed that profilin-1-induced accumulation of p27kip1 in breast cancer cells occurs most dramatically in the nuclear compartment and this is due to impaired protein degradation. Consistent with these observations, we found that phosphorylation of p27kip1 at T187 (a prerequisite for p27kip1 degradation by SCF-Skp2 complex in the nucleus) and S10 (a post-translational event that promotes nuclear export of p27kip1) residues are both significantly inhibited by profilin-1 overexpression. Elevating profilin-1 level suppresses AKT activation, and hyper-activating AKT through overexpression of a constitutively active mutant relieves profilin-1-induced inhibition of T187 phosphorylation and elevation of p27kip1. These data demonstrate that profilin-1 can control the cellular level of p27kip1 in breast cancer cells through affecting its post-translational modification in an AKT-dependent pathway. These findings may provide mechanistic insights underlying the tumor-suppressive action of profilin-1. Citation Format: Chang Jiang, William Veon, Partha Roy. Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1325. doi:10.1158/1538-7445.AM2014-1325

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥10μM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤1μM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1–15μM imatinib resulted in a dose dependent inhibition of 5-bromo-2′-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 in the nuclear compartment as a result of increased p27KIP1 protein stability. We demonstrate that the mechanism of p27KIP1 stabilization relied on inhibition of p27KIP1 phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.