Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.

Bone metastatic prostate cancer (PCa) promotes mesenchymal stem cell (MSC) recruitment and their differentiation into osteoblasts. However, the effects of bone-marrow derived MSCs on PCa cells are less explored. Here, we report MSC-derived interleukin-28 (IL-28) triggers prostate cancer cell apoptosis via IL-28 receptor alpha (IL-28Rα)-STAT1 signaling. However, chronic exposure to MSCs drives the selection of prostate cancer cells that are resistant to IL-28-induced apoptosis and therapeutics such as docetaxel. Further, MSC-selected/IL-28-resistant prostate cancer cells grow at accelerated rates in bone. Acquired resistance to apoptosis is PCa cell intrinsic, and is associated with a shift in IL-28Rα signaling via STAT1 to STAT3. Notably, STAT3 ablation or inhibition impairs MSC-selected prostate cancer cell growth and survival. Thus, bone marrow MSCs drive the emergence of therapy-resistant bone metastatic prostate cancer yet this can be disabled by targeting STAT3.

The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state.

Prostate cancer stem-like cells (PCSCs) are not only enriched in the CD44+PSA-/lo subpopulation but also employ androgen-independent signaling mechanisms for survival. CD44+ PCSCs defy androgen deprivation, resist chemo- and radiotherapy and are highly tumorigenic. Human prostate tissue microarray (TMA) staining revealed an increased membranous staining of CD44 in the luminal compartment in higher grade G7-G9 tumors versus staining of the basal layer in benign hyperplasia. To uncover tyrosine kinase/s critical for the survival of the CD44+PSA-/lo subpopulation, we performed an unbiased screen targeting 87 tyrosine kinases with gene specific siRNAs. Among a subset of tyrosine kinases crucial for PCSC survival, was a non-receptor tyrosine kinase, ACK1/TNK2, a critical regulator of castration resistant prostate cancer (CRPC) growth. Consistently, activated ACK1 as measured by phosphorylation at Tyr284 was significant in the CD44+PSA-/lo population. Conversely, pharmacological inhibition by ACK1 inhibitor, (R)-9bMS mitigated CD44+PSA-/lo sphere formation, overcame resistance to radiation-induced cell death, induced significant apoptosis in PCSCs and inhibited CD44+PSA-/lo xenograft tumor growth in castrated mice suggesting dependency of PCSCs on ACK1 for survival. Thus, blockade of ACK1/TNK2 could be a new therapeutic modality to target recalcitrant PCSCs.

Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys609 in the zinc finger DNA binding domain of AR (acK609-AR) that not only allowed AR translocation but also galvanized a distinct global transcription program, conferring enzalutamide insensitivity. Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase-mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, (R)-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma.