Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer activity [version 1; peer review: 1 approved]

Objective: To review the mechanisms of anti-cancer activity of nelfinavir and other protein inhibitors (PIs) based on evidences reported in the published literature. Methods: We extensively reviewed the literature concerning nelfinavir (NFV) as an off target anti-cancer drug and other PIs. A classification of PIs based on anti-cancer mode of action was proposed. Controversies regarding nelfinavir mode of action were also addressed. Conclusions: The two main mechanisms involved in anti-cancer activity are endoplasmic reticulum stress-unfolded protein response pathway and Akt inhibition. However there are many other effects, partially dependent and independent of those mentioned, that may be useful in cancer treatment, including MMP-9 and MMP-2 inhibition, down-regulation of CDK-2, VEGF, bFGF, NF-kB, STAT-3, HIF-1 alfa, IGF, EGFR, survivin, BCRP, androgen receptor, proteasome, fatty acid synthase (FAS), decrease in cellular ATP concentration and upregulation of TRAIL receptor DR5, Bax, increased radiosensitivity, and autophagy. The end result of all these effects is slower growth, decreased angiogenesis, decreased invasion and increased apoptosis, which means reduced proliferation and increased cancer cells death. PIs may be classified according to their anticancer activity at clinically achievable doses, in AKT inhibitors, ER stressors and Akt inhibitors/ER stressors. The article by Koltai reviews the extensive literature concerning Nelfinavir as an anti-cancer drug. Overall the study makes a good update of the evidences of nefilnavir anti-cancer activity and its role in controlling ERS-UPR pathway. It would be interesting to include in the discussion information about the different trials that are currently carry on with Nelfinavir in different tumors. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Introduction
In March 1997, the United States Food and Drug Administration (FDA) approved Nelvinavir (NFV, brand name Viracept) for HIV treatment in humans 1 . NFV is a safe, orally available, and potent drug against HIV-1 and HIV-2 2 . This protease inhibitor (PI) was developed by the private pharmaceutical sector and was a big success in the treatment of AIDS in association with other anti-retroviral drugs 3 . The introduction of PIs combined with HIV reverse transcriptase inhibitors started the era of HAART (highly active anti-retroviral treatment) and is nowadays the standard of care in HIV-AIDS 4 .
PIs inhibit HIV-1 and HIV-2 proteases (which are aspartate proteases), impeding virus replication and release of infecting viral particles from diseased cells. The mechanism of action of protease inhibitors involves competitive binding to the enzyme 5 .
NFV is being progressively displaced from HIV therapeutics by second generation HIV PIs, but has shown interesting off target actions in cancer.
The possible use of anti-HIV drugs against cancer is not new: in the 1990s AZT (zidovudine or azidothymidine) was proposed as antineoplastic drug, but clinical trials did not confirm the preliminary good results obtained in vitro 6 .
That HIV PIs target other molecules besides the HIV protease is quite evident if we examine adverse effects like insulin resistance and lipodystrophy. These and other evidences such as inhibition of tumor cell production of cytokines, anti-angiogenesis, induction of apoptosis and others, suggest off targets effects for PIs, and hints to the concept of a new class of drugs against cancer with multiple anti-cancer effects 6 . NFV, the most important anti-cancer drug of the PI family, if repurposed for cancer treatment, would have an important advantage: it has been used for more than 15 years in HIV treatment and its safety, pharmacokinetics, and adverse events are well known. Serious adverse events are not common with the exception of diarrhea when used at high doses.
Research on NFV as a potentially useful drug for cancer treatment 6 started in 2009.
In this article, we thoroughly review the literature published in this matter and analyze mainly the anti-cancer mechanisms of action of NFV.
Certain controversies regarding NFV activity in lipid metabolism will be considered in depth.

Evidences of nelfinavir anti-cancer activity
A partial response of Kaposi's sarcoma patients to PIs was published in 1998 7 and good results with regression (six complete responses out of 10 patients) 8 . In 1999 Niehues et al. 9 published complete regression of Kaposi's sarcoma in a child treated with highly active anti-retroviral therapy (HAART). Sgadari et al. (2003) described also the inhibition of Kaposi's sarcoma with protease inhibitors and they also mention that these drugs can antagonize vital properties of tumor cells like growth, invasion, tissue remodelling, angiogenesis and survival. They consider these effects to be a consequence of inhibition of invasion, matrix metalloprotease, proteasome and NF-kB signaling 10 . The possible mechanisms of PIs off target activity on tumor cells were described by pioneering work of Schmidtke et al. in 1999 11 : they observed that ritonavir was a modulator of proteasomal activity, allowed normal proliferation when used at low concentrations, but affected protein degradation when present at higher concentrations, and cell cycle was arrested.
Ikezoe et al. 12 described that protease inhibitors increased cellular growth inhibition of all transretinoic acid (ATRA) on cell cultures of myelocitytic leukaemia lines. Protease inhibitors also increased differentiation of acute myeloid leukemia cell lines.
In 2004, Ikezoe 13 described the mechanisms involved in anti-cancer activity of protease inhibitors in myeloma cells.
The mechanisms involved in PIs anti-cancer activity are summarized in chronological order on Table 1.

Nelfinavir and the ERS-UPR pathway
NFV inhibits the proteases S1P and S2P that are involved in SREBP-1 maturation and other proteases necessary for protein maturation and folding (yet not fully identified) in the endothelial reticulum 49 .
Activation of the unfolded protein response (UPR) starts in the ER when abnormal accumulation of protein is detected 57 . This was investigated thoroughly in yeasts where detection of abnormal protein occurs through Ire1p/Ern1p-mediated signaling from the ER (in mammals there are three sensor proteins IRE1α, PERK and ATF6 58 ). UPR activation leads to the specific removal of 252 nucleotides intron from a precursor mRNA of the transcription factor HAC-1p, and the resulting mature mRNA HAC-1p is translated to produce active HAC-1p. This transcription factor translocates to the nucleus and promotes the transcription of chaperones like GRP78 that facilitates removal of abnormal proteins from the ER through retrotranslocation and final disposal by the ubiquitin-proteasome pathway 59 .
HAC1 precursor mRNA is constitutively expressed but not translated until Ire1p/Ern1p sensor removes the necessary nucleotides. Thus the UPR is an intracellular signaling pathway where the ER "informs" the nucleus on the need to increase the levels of molecular chaperones and folding enzymes in order to maintain the ER homeostasis. Therefore UPR keeps unfolded proteins in the ER until they are correctly folded before they can go to their final destination. NFV seems to produce cellular stress by accumulation of misfolded or abnormal proteins in the ER, overwhelming the normal ER protein folding machinery 60 . Chaperones bound to unfolded proteins in the ER initiate protein kinase cascades that inhibit translation, reverse translocation, activate ubiquitination enzymes, induce autophagia, and when stress is extreme, induce apoptosis.

Study performed in Results
Jiang Saquinavir showed anti-proliferative and anti-invasive activity which was increased by the association with imatinib. Activation of NF-kB was decreased. Saquinavir also exerted a pro-apoptotic activity on NB lines, which was significantly increased by the association with imatinib.
Ismail, 2013 54 Hamster ovary cells Inhibits proximal insulin receptor signalling which may explain insulin resistance.
Escalante, 2013 55 Myeloma cells in culture and mice xenografts.
NFV is a calpain blocker. This activity enhances bortezomib (a proteosomal inhibitor) citotoxicity in vivo and in vitro. Drugs that block the HIV1-associated aspartyl protease also show cross reactivity with the cysteine protease calpain.
Bociaga-Jasik, 2013 56 Pre-adipocytes and adipocytes in culture Saquinavir decreased mitochondrial membrane potencial and intracellular ATP in adipocytes.    Figure 4. SREBP is synthesized as an ER transmembrane protein and transported to the Golgi upon appropriate stimulus. For activation of SREBP it is necessary that luminal S1P (a protease) cleaves first, followed by intramembrane S2P (another protease) to liberate the transcriptionally active amino-terminal segments of nSREBP. NFV inhibits S1P and S2P, so that transcriptionally active SREBP is not produced. Accumulation of inactive SREBP is one of the UPR initiators.  **It is necessary to underscore the finding that cancer stem cells expressing embryonic genes like Oct4, Sox2 and others, are particularly prone to apoptosis when PIs are used, particularly iopinavir (nelfinavir and saquinavir are also effective in this matter).

Interactions of NFV and PIs with other drugs
Indinavir and NFV increase anti-malarial action of artemisinin in vitro on Plasmodium falciparum 78 , but artemisinin has also an off target anti-cancer activity. Thus it is reasonable to raise the question: may the association of artesiminin with NFV increase anti-cancer activity?
Another research team 70 has included both, NFV and artesiminin, in a multidrug repurposed protocol (CUSP 9) for the treatment of relapsed glioblastoma.
Celecoxib is an ER stressor that may enhance NFV anti-tumor activity 79 .
Chloroquine and hydroxicloquine are autophagy inhibitors and may work synergistically with NFV, downregulating autophagy and increasing apoptosis 70,80 .
Nelfinavir may produce overproduction of mcl1 through upregulation of Erk ½, which would reduce apoptosis. The problem can be solved adding sorafenib 81,82 .
In breast cancer cells, tamoxifen enhances anti-cancer activity of NFV 83 . This synergism was independent of the estrogen receptor status so that the authors consider that the association of NFV and tamoxifen may be advantageous even in patients with no hormone responsive tumors.
Saquinavir has an interesting off target effect: it decreases intracellular ATP in adipocytes 56 . If this effect is similar in tumor cells, an association with metformin and 2-deoxyglucose may produce anticancer activity 84-86 . There is growing interest on metabolic perturbators in cancer therapy and saquinavir may play a role in this field.

Clinical trials
A phase I dose escalation trial performed in 2014 87 established a MTD (maximum tolerated dose) of 3125 mg twice daily and described that 45% of patients with solid tumors treated with this dose decreased AKT activity and increased ERS indicators. This indicated a possible benefit in neuroendocrine tumor patients and also established that dose limiting toxicity consists in neutropenia.
The dose (3125 mg bid) is more than twice the dose used in HIV treatment. But lower doses, in the range of those used in HIV treatment have been tested, combining nelfinavir with chemoradiotherapy in pancreatic cancer with evidence of efficacy 88 . No control group was used in this research, so comparison was established with known data from previous publications, mainly the favourable possibility of tumor resection after treatment.
Even lower doses (625 mg and 1250 mg bid) were tested in a phase I trial of NSCLC in stages IIIA/IIIB combined with chemoradiotherapy 89 . In nine out of 12 patients a PET scan was available posttreatment with 100% overall response (56% complete response and 44% partial response). Unfortunately, in this trial there was no control group; 50% of the patients (six out of 12) lived for more than 22 months after treatment; 25% (three out of 12) lived without disease for more than 32 months. The results may be considered favourable, even without a control group.
Buijsen et al. 75 recommend a dose of 750 mg NFV bid for a phase II trial of this drug in combination with chemoradiotherapy in locally advanced rectal cancer.
The first results of clinical trials did not show a meaningful improvement in the outcome of patients with refractory adenoid cystic carcinoma which is a malignant salivary gland tumor which usually has a poor prognosis. In this case NFV was used as monotherapy 76 .

Why nelfinavir has a role to play in cancer therapy?
Akt activation is an important step in cancer phenotype and is a key player in acquisition and maintenance of cancer hallmarks. Akt is a nodal regulator of cellular survival pathways 90 . There are no drugs at the present time that can inhibit this protein with a good safety profile. Wortmannin, perifosine and other chemicals designed for PI3K/Akt inhibition were too toxic for clinical use or have shown disappointing results, so they did not enter the medical practice. Insulin stimulation of Akt phosphorylation was reduced by 55% at achievable doses 20 . At the same time there is clear evidence that it favours apoptosis and growth inhibition at clinically tolerable and achievable doses.
This anti-Akt activity of NFV can be reinforced by concomitant mTOR inhibition which results in synergistic cytotoxicity 63 . This may be due to the fact that mTOR inhibition without Akt inhibition eliminates a negative biofeedback loop on Akt, producing increased phoshorilation of Akt 91 . According to Sarbassov 92 this negative feedback is born in the mTORC2 complex.
According to Carracedo 93 , this negative feedback loop goes as far as PI3K (Figure 6). mTOR inhibitors have become a new and important tool against cancer, for example in renal cell carcinoma. But the negative biofeedback loop on Akt must be solved to achieve really good results. NFV could be the clue.
But the most important anti-tumor activity of NVR is not limited to Akt inhibition but ER stress and UPR which may be one of the pathways leading to apoptosis 94 .
Additional features of NFV and other protease inhibitors are 1) the ability to sensitize cancer cells to chemoradiotherapy, 2) anti-angiogenesis by decreasing VEGF/HIF expression,  10) Down-regulation of MMP-9 (reduced expression and secretion of MMP-9 by human preadipocytes) 64,101 , 11) Increased apoptosis by NFV when associated with antiautophagy drugs like chloroquine or hydroxychloroquines, particularly in triple negative breast cancer cells 102 .

Possible controversies
The SREBP pathway for regulation of fat metabolism is initiated through proteolytic cleavage of precursor forms of the SREBPs (125 Kd protein) in ER membranes. When cells are in need of sterol, the precursor SREBPs are hydrolyzed by a 2-step mechanism involving membrane-bound serine protease S1P and a metalloprotease S2P. The N-terminal fragment of SREBP (nSREBP) is a 68 Kd protein that translocates to the nucleus where it works as a promoter-enhancer, binding to sterol regulatory elements located in DNA and activates gene transcription ( Figure 4). The nuclear SREBP can be rapidly degraded by a proteasome-mediated mechanism. This provides regulation of gene transcriptional activities 103 .
Transgenic mice over-expressing the constitutively active nuclear forms of the SREBPs (nSREBPs) revealed that overexpression of SREBP-1 or SREBP-2 leads to activation of genes involved in the cholesterol and fatty acid biosynthesis cascades. These transgenic mice displayed the classical features of generalized lipodystrophy, similar to those found in patients under PI therapy 104 .
Riddle et al. in 2001 105 found that PI therapy (they used ritonavir) induced the accumulation of activated SREBP-1 and SREBP-2 in the nucleus of liver and adipose tissues. As a consequence, fatty acid and cholesterol biosynthesis were increased in these tissues. The authors consider that lipodystrophy, hyperlipidemia, and insulin resistance, are the consequence of activated SREBP-1 and SREBP-2 accumulation in the nucleus of liver and adipose tissues. The possible mechanism for these events, according to their criteria is PI suppression of activated SREBP degradation in the nucleus. In summary, Riddles's study showed that ritonavir induced lipid metabolism abnormalities through stabilization of activated SREBP-1 and SREBP-2 in the nucleus of liver and adipose tissues.
These findings are in contrast with those of Guan 49,106 where NFV inhibited the nuclear translocation of the sterol regulatory element binding protein-1 (SREBP-1) in castration resistant prostate cancer and liposarcoma through inhibition of S1P. This led to accumulation of unprocessed SREBP-1. The controversy may be explained in the following way:
2) SREBP-1a and -1c have different expression profiles: SREBP-1a is highly expressed in proliferating cells, such as cancer cells, while SREBP-1c is the predominant form in normal cells, particularly hepatocytes 104 .
3) The target genes for the three SREBP isoforms are different. May this difference be due to tissue-specific effects of NFV? Does NFV have different effects in tumor tissues and normal tissues?
To definitely solve these controversies, it is necessary to proceed with further experimental research, but the findings described above necessarily raise the doubt that mechanisms that work in tumor cells might be slightly different from those working in hepatocytes and adipocytes.

Possible negative aspects of PIs in cancer
Despite the anti-cancer activity of NFV and PIs, these drugs do not reduce the risk of developing cancer in HIV population 108 and also exert certain depression of immunological functions, interfering with the differentiation program of monocytes into dendritic cells 109 .
PIs increase the expression of P-glycoprotein (ABCB1) in Kaposi's sarcoma cell lines increasing the multidrug resistance phenotype 110 .
At the same time ABCB1 expression depends on Akt activation 111 and NFV inhibits partially Akt. The final result of the two antagonistic aspects requires further research.
There are well known undesirable side effects with HIV PIs, like hyperlipidemia, insulin resistance and lypodystrophy (peripheral fat wasting and excessive central fat deposition). One of the main responsible mechanisms of these side effects is the suppression of the breakdown of SREBP in the liver and adipose tissues resulting in increased fatty acid and cholesterol biosynthesis. SREBP accumulation in adipose tissue causes lipodystrophy.
PIs suppress proteasome-mediated breakdown of nascent apolipoprotein (apo) B, resulting in the overproduction of triglyceride. Finally, PIs also suppress the inhibition of the glucose transporter GLUT-4 activity in adipose tissue and muscle. This contributes directly to insulin resistance and diabetes 112 .
Hepatomegaly and hepatic steatosis are direct consequences of the metabolic alterations explained above 105 .

New PIs with anti-cancer activity
In 2010 You et al. 113 synthesized a new indinavir analogue with remarkable anti-cancer activity, similar to NFV: CH05-10. This drug achieved similar cytotoxity to NFV but at lower concentrations, against leukaemia, melanoma, ovarian and prostate cancer cell lines.
In 2009 Saquinavir-NO was introduced 114 ; it showed interesting anti-cancer properties in melanoma xenografts with significantly lower toxicity than saquinavir.

Conclusions
The most relevant mechanisms of PIs anti-cancer activity are Akt inhibition and ER stress.
Following our exhaustive analysis of the current medical literature we conclude that NFV anti-cancer activity is mainly dependent on ER stress-UPR.
Akt inhibition plays also a very important role but is not the unique or main source of anti-cancer effects.
The evidences that support these conclusions are: 1) Even at very high doses of NFV (3,125 mg bid), Akt achieved a level of inhibition around 55% in cell culture 20 .
2) NFV is at the same time a strong ER stressor and an Akt inhibitor.
3) Anti-cancer activity can be achieved at much lower doses than those necessary for Akt inhibition. There is enough evidence of NFV anti-cancer effects and there is adequate knowledge of how this activity works, so that NFV deserves well designed phase II clinical trials, as adjunct cancer therapy.
Associations with proteasomal inhibitors, celecoxib and other cell stressor should also be investigated in the clinical setting due to possible synergy. Tamoxifen with NFV may show interesting results in breast cancer.
Although a large amount of publications, including reviews, have been written on NFV and other PIs in cancer, none has been dedicated to a thorough examination and analysis of the mode of action of these pharmaceuticals as off target drugs (with the exception of the review by Gantt et al. 2 ). It is hoped that this review will encourage an increment adequately powered and well-designed clinical trials in the various cancer types, beyond the phase I trials that have been recently performed, and specifically trials where these compounds may be tested in association with other known anti-cancer pharmaceuticals like NFV associated to bortezomib and hydroxychloroquine in myeloma, or mTOR inhibitors with NFV in HNSCC and many other possible combinations where the dual feature of NFV, ER stressor and Akt inhibitor, are required.
In myeloma NFV increases proteasome inhibition by bortezomib and may overcome resistance to proteasomal inhibitors. This is an action exclusive of NFV and not shared with other PIs 67 , with the additional advantage that NFV shows the highest cytotoxic activity against primary myeloma cells.
If apoptosis is described as a cascade, then apoptosis stimulator drugs like NFV should be viewed as enhancers of this cascade. An initiator of the cascade is still necessary, for example chemoradiotherapy. After this initial step, apoptosis stimulator drugs increase the amount of cells entering this pathway. This might be one of possible reasons why nelfinavir alone has shown poor results in a clinical trial used as monotherapy.
This does not mean that NFV cannot act as an initiator, but the evidences show that it is prone to be an enhancer of apoptosis rather than an initiator.

Future directions
All the evidences presented in this review reinforce the concept that NFV is a useful drug in cancer treatment. It should be considered in association with chemoradiotherapy in the design of new protocols for diseases like multiple myeloma (in association with bortezomib and hydroxychloroquine) and prostate, pancreas and lung cancer where clinical trials are ongoing. New PIs are being developed with better anti-cancer profile like CH05-10 and saquinavir-NO 77 and further development of new PIs with stronger anti-cancer activity, will probably go on in the future.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work.