Mitchell E. Menezes

Dickkopf1: A tumor suppressor or metastasis promoter?

Dickkopf1 (DKK1), a secreted inhibitor of the Wnt/β‐catenin pathway, is a negative regulator of bone formation. DKK1 acts as a switch that transitions prostate cancer bone metastases from osteolytic to osteoblastic and also is an active indicator of poor outcome for multiple myeloma. However, in other tumor types, DKK1 upregulation or overexpression suppresses tumor growth. Thus, the role of DKK1 in cancer appears to be diverse. This raises a question: Could the increased levels of DKK1 still be tumor protective when observed in high levels in the serum of patients? Here, we summarize the diverse, seemingly contradicting roles of DKK1 and attempt to explain the apparent dichotomy in its activity. We propose that DKK1 is a critical secreted factor that modulates microenvironment. Based on the location and components of the microenvironment DKK1 will support different outcomes.

DNAJB6 Induces Degradation of β-Catenin and Causes Partial Reversal of Mesenchymal Phenotype

We showed that expression of MRJ (DNAJB6) protein is lost in invasive ductal carcinoma, and restoration of MRJ(L) restricts malignant behavior of breast cancer and melanoma cells. However, the signaling pathways influenced by MRJ(L) are largely unknown. Our observations revealed that MRJ(L) expression causes changes in cell morphology concomitant with down-regulation of several mesenchymal markers, viz. vimentin, N-cadherin, Twist, and Slug, and up-regulation of epithelial marker keratin 18. Importantly, MRJ(L) expression led to reduced levels of β-catenin, an epithelial mesenchymal transition marker, and a critical player in the Wnt pathway. We found that MRJ(L) up-regulates expression of DKK1, a well known Wnt/β-catenin signaling inhibitor, that causes degradation of β-catenin. Re-expression of DNAJB6 alters the Wnt/β-catenin signaling in cancer cells, leading to partial reversal of the mesenchymal phenotype. Thus, MRJ(L) may play a role in maintaining an epithelial phenotype, and inhibition of the Wnt/β-catenin pathway may be one of the potential mechanisms contributing to the restriction of malignant behavior by MRJ(L).

DNAJB6 governs a novel regulatory loop determining Wnt/β-catenin signalling activity.

DKK1 (dickkopf 1 homologue) is a secreted inhibitor of the Wnt signalling pathway and a critical modulator of tumour promotion and the tumour microenvironment. However, mechanisms regulating DKK1 expression are understudied. DNAJB6 {DnaJ [HSP40 (heat-shock protein 40 kDa)] homologue, subfamily B, member 6} is an HSP40 family member whose expression is compromised during progression of breast cancer and melanoma. Inhibition of the Wnt/β-catenin signalling pathway by up-regulation of DKK1 is one of the key mechanisms by which DNAJB6 suppresses tumour metastasis and EMT (epithelial-mesenchymal transition). Analysis of the DKK1 promoter to define the cis-site responsible for its up-regulation by DNAJB6 revealed the presence of two binding sites for a transcriptional repressor, MSX1 (muscle segment homeobox 1). Our investigations showed that MSX1 binds the DKK1 promoter and inhibits DKK1 transcription. Interestingly, silencing DNAJB6 resulted in up-regulation of MSX1 concomitant with increased stabilization of β-catenin. ChIP (chromatin immunoprecipitation) studies revealed that β-catenin binds the MSX1 promoter and stabilization of β-catenin elevates MSX1 transcription, indicating that β-catenin works as a transcription co-activator for MSX1. Functionally, exogenous expression of MSX1 in DNAJB6-expressing cells promotes the mesenchymal phenotype by suppression of DKK1. Thus we have identified a novel regulatory mechanism of DNAJB6-mediated DKK1 transcriptional up-regulation that can influence EMT. DKK1 is a feedback regulator of β-catenin levels and thus our studies also define an additional negative control of this β-catenin/DKK1 feedback loop by MSX1, which may potentially contribute to excessive stabilization of β-catenin.

MDA-7/IL-24 functions as a tumor suppressor gene in vivo in transgenic mouse models of breast cancer

Melanoma differentiation associated gene-7/Interleukin-24 (MDA-7/IL-24) is a novel member of the IL-10 gene family that selectively induces apoptosis and toxic autophagy in a broad spectrum of human cancers, including breast cancer, without harming normal cells or tissues. The ability to investigate the critical events underlying cancer initiation and progression, as well as the capacity to test the efficacy of novel therapeutics, has been significantly advanced by the development of genetically engineered mice (GEMs) that accurately recapitulate specific human cancers. We utilized three transgenic mouse models to better comprehend the in vivo role of MDA-7/IL-24 in breast cancer. Using the MMTV-PyMT spontaneous mammary tumor model, we confirmed that exogenously introducing MDA-7/IL-24 using a Cancer Terminator Virus caused a reduction in tumor burden and also produced an antitumor “bystander” effect. Next we performed xenograft studies in a newly created MMTV-MDA-7 transgenic model that over-expresses MDA-7/IL-24 in the mammary glands during pregnancy and lactation, and found that MDA-7/IL-24 overexpression delayed tumor growth following orthotopic injection of a murine PDX tumor cell line (mPDX) derived from a tumor formed in an MMTV-PyMT mouse. We also crossed the MMTV-MDA-7 line to MMTV-Erbb2 transgenic mice and found that MDA-7/IL-24 overexpression delayed the onset of mammary tumor development in this model of spontaneous mammary tumorigenesis as well. Finally, we assessed the role of MDA-7/IL-24 in immune regulation, which can potentially contribute to tumor suppression in vivo. Our findings provide further direct in vivo evidence for the role of MDA-7/IL-24 in tumor suppression in breast cancer in immune-competent transgenic mice.

Loss of tumor suppressor Merlin results in aberrant activation of Wnt/β-catenin signaling in cancer

The expression of the tumor suppressor Merlin is compromised in nervous system malignancies due to genomic aberrations. We demonstrated for the first time, that in breast cancer, Merlin protein expression is lost due to proteasome-mediated elimination. Immunohistochemical analysis of tumor tissues from patients with metastatic breast cancer revealed characteristically reduced Merlin expression. Importantly, we identified a functional role for Merlin in impeding breast tumor xenograft growth and reducing invasive characteristics. We sought to determine a possible mechanism by which Merlin accomplishes this reduction in malignant activity. We observed that breast and pancreatic cancer cells with loss of Merlin show an aberrant increase in the activity of β-catenin concomitant with nuclear localization of β-catenin. We discovered that Merlin physically interacts with β-catenin, alters the sub-cellular localization of β-catenin, and significantly reduces the protein levels of β-catenin by targeting it for degradation through the upregulation of Axin1. Consequently, restoration of Merlin inhibited β-catenin-mediated transcriptional activity in breast and pancreatic cancer cells. We also present evidence that loss of Merlin sensitizes tumor cells to inhibition by compounds that target β-catenin-mediated activity. Thus, this study provides compelling evidence that Merlin reduces the malignant activity of pancreatic and breast cancer, in part by suppressing the Wnt/β-catenin pathway. Given the potent role of Wnt/β-catenin signaling in breast and pancreatic cancer and the flurry of activity to test β-catenin inhibitors in the clinic, our findings are opportune and provide evidence for Merlin in restraining aberrant activation of Wnt/β-catenin signaling.

MDA-9/Syntenin (SDCBP) modulates small GTPases RhoA and Cdc42 via transforming growth factor β1 to enhance epithelial-mesenchymal transition in breast cancer

Epithelial-mesenchymal transition (EMT) is one of the decisive steps regulating cancer invasion and metastasis. However, the molecular mechanisms underlying this transition require further clarification. MDA-9/syntenin (SDCBP) expression is elevated in breast cancer patient samples as well as cultured breast cancer cells. Silencing expression of MDA-9 in mesenchymal metastatic breast cancer cells triggered a change in cell morphology in both 2D- and 3D-cultures to a more epithelial-like phenotype, along with changes in EMT markers, cytoskeletal rearrangement and decreased invasion. Conversely, over expressing MDA-9 in epithelial non-metastatic breast cancer cells instigated a change in morphology to a more mesenchymal phenotype with corresponding changes in EMT markers, cytoskeletal rearrangement and an increase in invasion. We also found that MDA-9 upregulated active levels of known modulators of EMT, the small GTPases RhoA and Cdc42, via TGFβ1. Reintroducing TGFβ1 in MDA-9 silenced cells restored active RhoA and cdc42 levels, modulated cytoskeletal rearrangement and increased invasion. We further determined that MDA-9 interacts with TGFβ1 via its PDZ1 domain. Finally, in vivo studies demonstrated that silencing the expression of MDA-9 resulted in decreased lung metastasis and TGFβ1 re-expression partially restored lung metastases. Our findings provide evidence for the relevance of MDA-9 in mediating EMT in breast cancer and support the potential of MDA-9 as a therapeutic target against metastatic disease.

Cellular stress stimulates nuclear localization signal (NLS) independent nuclear transport of MRJ

HSP40 family member MRJ (DNAJB6) has been in the spot light for its relevance to Huntingtons, Parkinsons diseases, limb-girdle muscular dystrophy, placental development, neural stem cells, cell cycle and malignancies such as breast cancer and melanoma. This gene has two spliced variants coding for 2 distinct proteins with significant homology. However, MRJ(L) (large variant) is predominantly localized to the nucleus whereas MRJ(S) (small variant) is predominantly cytoplasmic. Interestingly MRJ(S) translocates to the nucleus in response to heat shock. The classical heat shock proteins respond to crises (stress) by increasing the number of molecules, usually by transcriptional up-regulation. Our studies imply that a quick increase in the molar concentration of MRJ in the nuclear compartment is a novel method by which MRJ responds to stress. We found that MRJ(S) shows NLS (nuclear localization signal) independent nuclear localization in response to heat shock and hypoxia. The specificity of this response is realized due to lack of such response by MRJ(S) when challenged by other stressors, such as some cytokines or UV light. Deletion analysis has allowed us to narrow down on a 20 amino acid stretch at the C-terminal region of MRJ(S) as a potential stress sensing region. Functional studies indicated that constitutive nuclear localization of MRJ(S) promoted attributes of malignancy such as proliferation and invasiveness overall indicating distinct phenotypic characteristics of nuclear MRJ(S).

Suppression of Her2/Neu mammary tumor development in mda-7/IL-24 transgenic mice.

Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) encodes a tumor suppressor gene implicated in the growth of various tumor types including breast cancer. We previously demonstrated that recombinant adenovirus-mediated mda-7/IL-24 expression in the mammary glands of carcinogen-treated (methylnitrosourea, MNU) rats suppressed mammary tumor development. Since most MNU-induced tumors in rats contain activating mutations in Ha-ras, which arenot frequently detected in humans, we presently examined the effect of MDA-7/IL-24 on Her2/Neu-induced mammary tumors, in which the RAS pathway is induced. We generated tet-inducible MDA-7/IL-24 transgenic mice and crossed them with Her2/Neu transgenic mice. Triple compound transgenic mice treated with doxycycline exhibited a strong inhibition of tumor development, demonstrating tumor suppressor activity by MDA-7/IL-24 in immune-competent mice. MDA-7/IL-24 induction also inhibited growth of tumors generated following injection of Her2/Neu tumor cells isolated from triple compound transgenic mice that had not been treated with doxycycline, into the mammary fat pads of isogenic FVB mice. Despite initial growth suppression, tumors in triple compound transgenic mice lost mda-7/IL-24 expression and grew, albeit after longer latency, indicating that continuous presence of this cytokine within tumor microenvironment is crucial to sustain tumor inhibitory activity. Mechanistically, MDA-7/IL-24 exerted its tumor suppression effect on HER2+ breast cancer cells, at least in part, through PERP, a member of PMP-22 family with growth arrest and apoptosis-inducing capacity. Overall, our results establish mda-7/IL-24 as a suppressor of mammary tumor development and provide a rationale for using this cytokine in the prevention/treatment of human breast cancer.

Abstract 2403: DNAJB6 suppresses the Wnt/β-catenin pathway

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnDNAJB6 is an HSP40 family protein which plays an important role in regulating tumorigenicity and metastasis of breast cancer and melanoma. Expression of DNAJB6 is found to be reduced in advanced stages of the disease. Restoration of its expression significantly decreases tumor growth and metastasis in xenograft studies. DNAJB6 expressing cells also show altered morphology, gain in epithelial markers and loss of mesenchymal markers and increased expression of DKK1, a secreted Wnt/β-catenin signaling. Our current studies elaborate the intricate mechanistic details about contributions of DNAJB6 to the negative regulation of the Wnt/β-catenin pathway. We describe the important role of the “J” domain of DNAJB6 in inhibiting Wnt/β-catenin. Deletion of “J” domain renders DNAJB6 incapable of impeding tumor growth and metastasis. Co-immunoprecipitation, mammalian-two-hybrid and FPLC studies show that DNAJB6 can form a multimeric complex with HSPA8 (HSC70), GSK3β and PP2A. DNAJB6 binds HSC70 and dephosphorylates GSK3β by recruiting PP2A. Activated GSK3β in-turn degrades β-catenin protein. Deletion of the J domain does not allow the formation of this multiprotein complex, which inactivates GSK3β and restores β-catenin levels. We further noted that the morphology of the cells grown in three dimensional matrix show distinct difference in WT-DNAJB6 and J-domain deleted expressors. In vivo and in vitro studies done with J domain deleted expressors showed increased malignancy. We also demonstrate the importance of J domain of DNAJB6 in suppressing osteopontin as one of the critical factors contributing to its reduced malignant activity in suppressing invasion and growth of xenografts. In summary, our studies unravel a novel mechanistic role of DNAJB6 in regulating GSK3β, a critical kinase which restricts tumor progression.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2403. doi:1538-7445.AM2012-2403

Abstract 997: Stability of β-catenin decides the tumor suppressive effect of MRJ

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnMammalian relative of DnaJ (MRJ), is a member of the heat shock protein 40 family. We have found that MRJ is lost in advanced grades of breast cancer. Functional studies showed that ectopic expression of the large spliced variant of MRJ, MRJ(L), results in reduced malignant properties as indicated by reduced motility, migration, invasion and soft agar colonization. Furthermore, xenograft studies in athymic mice showed that MRJ(L) expression reduced tumor growth and metastasis. Our investigations also revealed that Wnt/β-catenin signaling is one of the pathways impacted by MRJ(L) expression. Specifically, we found that MRJ(L) expression led to enhanced degradation of β-catenin. Our studies showed that MRJ(L) mediated its tumor suppressive effects by upregulation of DKK1, a negative regulator of Wnt/β-catenin signaling, which promotes proteosome mediated degradation of β-catenin. Hence, we hypothesized that introduction of a degradation-resistant form of β-catenin will lead to restoration of malignant attributes of the MRJ(L) expressing cells. S37A mutation of β-catenin has been known to render it insensitive to ubiquitination-mediated proteosomal degradation. We established stable clones co-expressing S37A β-catenin and MRJ(L). Our analysis of these clones showed that the TCF/LEF activity, as a measure of Wnt/β-catenin signaling, was elevated 10 fold, as measured by the increased activity of the luciferase reporter, TOPFlash. We also found that downstream transcriptional target of Wnt/β-catenin signaling, cyclin D1 showed increased expression in S37A β-catenin clones as compared to the MRJ(L) expressors. Synchronized cell cycle analysis demonstrated that cells co-expressing S37A β-catenin and MRJ(L) resulted in marked increase in S phase at 2 and 24 hours after the release of cell cycle block. The MRJ(L) expressors; did not go through an increase in the S phase in the 24 hour period after the release. Analysis of tumor growth in vivo (xenograft model) revealed that S37A β-catenin was able to restore the malignant activity overriding the suppressive effect of MRJ(L). Thus controlled degradation of β-catenin, brought about by MRJ(L), is an important event in maintaining cells in a non-tumorigenic state.nnAcknowledgement: We acknowledge grant support from the NCI: 1R01CA140472nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 997. doi:10.1158/1538-7445.AM2011-997